System and method for high resolution analysis of nucleic acids to detect sequence variations

ABSTRACT

Provided herein are methods for assaying a biological sample for microorganisms having drug resistant and/or drug sensitive phenotypes, wherein the methods are capable of detecting resistant and sensitive phenotypes associated with known and/or unknown mutations. In some aspects, methods are provided for detecting drug resistant  Myobacterium tuberculosis  (MTb), including multi-drug resistant MTb, wherein drug resistance is associated with one or more novel mutations. Also provided are systems, kits, and compositions related to such methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/058,637, filed on Mar. 28, 2008, which claims benefit under 35 U.S.C.§119(e) to U.S. Provisional Application No. 60/908,604 filed Mar. 28,2007, both of which are hereby incorporated by reference in itsentirety.

FIELD

The present invention relates generally to systems and methods forpreparing and analyzing biological samples to detect nucleic acidsequence variations associated with phenotypes of interest. In certainembodiments, methods and systems are provided for amplifying nucleicacids of microorganisms from a biological sample, and detecting sequencevariations associated with drug resistance and/or sensitivity.

BACKGROUND

Very few developments in the history of science have had such a profoundimpact upon human life as advances in controlling pathogenicmicroorganisms. It was not until the late 19^(th) and early 20^(th)centuries that the work of Pasteur and Koch established microorganismsas the cause of infectious diseases and provided strategies that led torational prevention and control strategies. The sulphonamides were amongthe first groups of compounds discovered to suppress microorganisminfections, and though little was known about their mechanism of action,the discovery stimulated a massive hunt for more effective antibioticcompounds. The isolation of an impure but highly active preparation ofpenicillin by Florey and Chain in 1940, and the subsequent success ofpenicillin diverted additional scientific effort towards the search forantibiotics, leading to the discovery of approximately 3,000 namedantibiotics. However, despite rapid progress in the discovery of newcompounds, only 50 of the named antibiotics have met with clinical use,and even fewer are commonly used in treating microorganism diseases.

The initial effectiveness of antibiotics against microorganisminfections has been partly offset by the emergence of strains ofmicroorganisms that are resistant to various antibiotics. Antibioticresistance has proven difficult to overcome because of the acceleratedevolutionary adaptability of microorganisms, the increasing overuse ofantibiotics in the clinic, and lack of patient compliance in completingprescribed dosing regimens. Resistance issues have made many otherwisecurable diseases, such as gonorrhea and typhoid, difficult to treat. Inaddition, microorganisms resistant to vancomycin, one of the lastbroadly effective antibiotics, are becoming increasingly prevalent inhospitals.

New antibiotic compounds are constantly being developed to keepinfectious microorganisms at bay, and an understanding of the mechanismsof antibiotic resistance has proven valuable in the development process.Advances in genomics allow researchers to identify biochemical pathwaysthat are susceptible to inhibition or modification, and to rationallydesign drugs targeted against such pathways. Many drugs exert atherapeutic effect by binding to a microorganism protein and modifyingits structure and/or function. In such cases, microorganisms can developimmunity by physical modification of the target protein in a manner thatinterferes with drug binding or activity. For example, resistance to theantibiotic erythromycin in several microorganisms results from avariation of the 50S ribosome subunit that causes a reduced affinity ofribosomes for erythromycin. Since a protein's structure/function isdetermined by its primary sequence, which is in turn determined by thesequence of the nucleic acid encoding the protein, nucleic acid sequencevariations associated with drug resistant phenotypes are usefuldiagnostic indicators of drug resistance.

While methods have been established to identify nucleic acid sequencevariations in microorganisms, existing techniques are limited by therequirement for foreknowledge of the particular mutations or othervariations being used as diagnostic indicators. As a result, knownscreening procedures often overlook newly developed and/oruncharacterized sequence variations associated with drug resistance orother characteristics of interest.

Accordingly, there is a need in the art for fast, affordable, andreliable methods for detecting both known and unknown nucleic acidsequence variations having diagnostic utility, including mutationsassociated with drug sensitivity and/or drug resistance patterns in awide variety of organisms, such as yeasts, viruses, fungi, bacteria,parasites and even humans.

SUMMARY

In some aspects, methods are provided for determining the responsivenessof a microorganism to a drug, the methods comprising obtaining abiological sample from a patient, the sample containing an infectiousmicroorganism; amplifying one or more segments of DNA of themicroorganism, the one or more segments including at least onepolymorphism associated with responsiveness of the microorganism to adrug of interest; and assaying the one or more amplified DNA segmentsfor sequence variations relative to a reference sequence, wherein avariation in one or more of the amplified DNA segments indicatesresponsiveness of the microorganism to the drug.

In some preferred embodiments, amplified DNA is assayed for sequencevariations using high resolution melting curve analysis. In variousembodiments, melting curve analysis involves incubating the amplifiedDNA (target DNA) with a complementary reference sequence, such as awild-type sequence, in the presence of a DNA-binding fluorescent dyethat emits a substantially different level of fluorescence in thepresence of double-stranded DNA (dsDNA) relative to single-stranded DNA(ssDNA). In some preferred embodiments, the DNA-binding dye isdsDNA-specific dye, such as SYBR Green I or SYBR Green II, and meltingcurve analysis involves monitoring the level of fluorescence as afunction of time as the assay solution is slowly heated at a constantrate. Advantageously, melting curve analysis according to methodsprovided herein can accurately detect single base pair mismatchesbetween a target DNA sequence and a reference sequence, and/ormismatches in two, three, four, five, or more bases.

In some embodiments, the reference sequence used in melting curveanalyses of methods provided herein includes at least one polymorphismassociated with drug responsiveness, such as drug resistance or drugsensitivity, and the analysis detects one or more additionalpolymorphisms in the DNA segment that includes the polymorphismassociated with drug responsiveness.

In some aspects, methods are provided for determining if a patient isamenable to treatment with a drug, the methods comprising obtaining abiological sample from a patient, where the sample contains Myobacteriumtuberculosis (MTb); amplifying one or more segments of MTb DNA of SEQ IDNOS: 142-204, each of the one or more segments including at least onepolymorphism associated with sensitivity of the MTb to an antibioticdrug; and assaying the one or more amplified DNA segments for sequencevariations relative to the corresponding sequence among SEQ ID NOS:142-204, wherein a variation in one or more of the amplified DNAsegments indicates sensitivity of MTb to the antibiotic drug. In someembodiments, variations in two or more of the amplified DNA segmentsindicates sensitivity of MTb to the antibiotic drug.

In some embodiments, the MTb DNA of SEQ ID NOS: 142-204 is amplified byPCR using the corresponding primers of SEQ ID NOS: 11-136.

In various embodiments, amplified MTb DNA comprises one or more of SEQID NOS: 142-145, and a variation in one or more of the amplified DNAsegments indicates sensitivity of MTb to rifampicin; the amplified MTbDNA comprises one or more of SEQ ID NOS: 146-151, and a variation in oneor more of the amplified DNA segments indicates sensitivity of MTb topyrazinamide; the amplified MTb DNA comprises one or more of SEQ ID NOS:152-154, and a variation in one or more of the amplified DNA segmentsindicates sensitivity of MTb to streptomycin; the amplified MTb DNAcomprises one or more of SEQ ID NOS: 155-176, and a variation in one ormore of the amplified DNA segments indicates sensitivity of MTb toisoniazid; the amplified MTb DNA comprises one or more of SEQ ID NOS:177-198, and a variation in one or more of the amplified DNA segmentsindicates sensitivity of MTb to ethambutol; the amplified MTb DNAcomprises one or more of SEQ ID NOS: 199-203, and a variation in one ormore of the amplified DNA segments indicates sensitivity of MTb to oneor both of capreomycin and viomycin; and/or the amplified MTb DNAcomprises SEQ ID NO: 204; and a variation in the amplified DNA segmentindicates sensitivity of MTb to one or more of oxifloxacin,moxifloxican, gatifloxican, sitafloxacin, ofloxacin, levofloxacin, andsparfloxacin.

In an additional aspect, kits are provided for determining whether apatient is amenable to treatment with a drug, where the kits comprise atleast one primer pair of SEQ ID NOS: 1-136; at least one nucleotideprobe complementary to an amplicon of SEQ ID NOS: 137-204; andinstructions for using the at least one primer pair to amplify DNA froma biological sample of a patient infected with Myobacterium tuberculosis(MTb), and using the at least one nucleotide probe to detect sequencevariations within the amplified DNA using high resolution melting curveanalysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A graphical representation of a design for overlapping primerannealing temperatures and template denaturation temperatures.

FIG. 2: An illustration of conventional amplification products by realtime PCR.

FIG. 3: A graph showing high temperature PCR amplification of the sametemplate used in FIG. 2.

FIG. 4: Graph showing the HTPCR amplification of the same templatematerial using different starting material concentrations

FIG. 5: A—Comparison of HTPCR products from the CFP32 gene of M.tuberculosis in water and MycoBuffer. B—Comparison of HTPCR productsfrom the IS6110 gene region of M. tuberculosis in water and MycoBuffer.C—Comparison of HTPCR products from the btMTb gene region of M.tuberculosis in water and MycoBuffer. D—Comparison of HTPCR productsfrom the IS6110 Transposase target gene region of M. tuberculosis inwater and MycoBuffer. E—Comparison of HTPCR products from the BTTb generegion of M. tuberculosis in water and MycoBuffer.

FIG. 6: Graphical representations of the amplification products from aRifampicin resistance screen. A—Homodplex and heteroduplex amplificationproducts. B—Melting curves of the homo- and heteroduplex products.C—Difference plot between the melting curves in B.

FIG. 7: Graphical representation of the curve analysis and differencecurves of control and sensitive samples.

FIG. 8: Graphical representation showing different fluorescent curvesfor different nucleic acids.

FIG. 9: Graphical representation of difference curve analysis betweencontrol, resistant and sensitive samples.

FIG. 10A: Difference curve analysis of Rifampicin sensitive andresistant samples from M. tuberculosis. B. Difference curve analysis ofStreptomycin sensitive and resistant samples from M. tuberculosis.

FIG. 11: Difference curve analysis of Terbinafine resistant samples fromS. cerevisiae.

FIG. 12: Difference curve analysis of Taxane sensitive and resistantsamples from humans.

FIG. 13: Difference curve analysis of chloroquine resistant samples fromMalaria infections.

FIG. 14: Difference curve analysis of Zidovudine sensitive and resistantsamples from HIV.

FIG. 15: Difference curve analysis of Vancomycin sensitive and resistantsamples from S. aureus.

FIG. 16: Agarose gel analysis of M. tuberculosis DNA products amplifiedby dynamic flux amplification simulation.

FIG. 17: Real time analysis of S. tymphimurium DNA amplificationproducts by dynamic flux amplification.

DETAILED DESCRIPTION OF ILLUSTRATIVE ASPECTS

Provided herein are reliable, low-cost methods for detecting nucleicacid sequence variations associated with one or more phenotypiccharacteristics having diagnostic utility in the treatment of a disease,disorder, or condition. In some aspects, methods described herein areuseful for detecting nucleic acid sequence variations associated withthe responsiveness of a microorganism to one or more drugs. Alsoprovided herein are compositions, systems, and kits related to theinstant methods. While a number of aspects and advantages of the instantinvention are described herein with respect to various methods, skilledartisans will recognize that such aspects and advantages are alsoapplicable to related compositions, systems, kits, and the like.

The term “microorganism” as used herein can refer to bacteria, fungi,protozoa, parasites and/or viruses. In various preferred embodiments,the microorganism is a bacterial pathogen. In some preferredembodiments, the microorganism is Mycobacterium tuberculosis. However,while a number of aspects and advantages of the instant invention aredescribed herein in relation to Mycobacterium tuberculosis, skilledartisans will recognize that such aspects and advantages are alsoapplicable for other microorganisms, and for a variety of diseases andconditions. Non-limiting examples of microorganisms useful in thediagnostic methods provided herein are set forth in Table I, along withvariable sequence elements related to the drug responsiveness of suchmicroorganisms.

The “subject” referred to herein can be any organism capable of hostinga microorganism, including but not limited to, experimental animals(e.g., mice, rats, rabbits, and the like) and humans. In variouspreferred embodiments, the subject is a human patient suffering from aninfectious disease. In some preferred embodiments, the patient suffersfrom tuberculosis.

A “biological sample” described herein can include any biologicalmaterial taken from a subject, including but not limited to,expectorations (e.g., sputum), blood, blood cells (e.g., lymphocytes),tissue, biopsies, cultured cells, pleural, peritoneal, or cerebrospinalfluid, sweat, feces, and urine. In some embodiments, a biological samplefrom a subject is treated, e.g., to culture an infectious microorganismand/or amplify its genetic material, before being assayed according tomethods provided herein.

As used herein, the term “drug” can refer to any compound, agent,treatment modality, or combination thereof. In some preferred aspects,the drug is an antibiotic compound.

The term “target nucleic acid(s)” as used herein refers to nucleic acidsderived from an infectious microorganism, as distinguished from nucleicacids of the subject and/or foreign nucleic acids unrelated to thedisease, disorder, or condition intended for treatment. In some aspects,a target nucleic acid is a nucleic acid of a microorganism that isassayed according to a method provided herein.

The term “reference nucleic acid” as used herein refers to a nucleicacid corresponding to a target nucleic acid (e.g., representing the sameportion of genomic DNA), that differs from the target nucleic acid byone or more sequence variations. For example, in some aspects, areference nucleic acid has the sequence of a wild-type microorganism(e.g., with respect to responsiveness to a drug of interest). In furtheraspects, a reference nucleic acid has the sequence of a wild-type humancell, such as a diseased cell, including, e.g., a human cancer cell.

The term “sequence variation” as used herein in relation to nucleicacids refers to a difference in the sequence of a nucleic acid relativeto the sequence of a corresponding nucleic acid (e.g., a sequencerepresenting the same gene or other portion of genomic DNA). In somepreferred embodiments, sequence variations detected according to variousmethods provided herein are “Single Nucleotide Polymorphisms” (“SNPs”),resulting from a difference in the identity of a single nucleotidebetween a target nucleic acid and a reference nucleic acid. In furtherembodiments, sequence variations detected according to various methodsprovided herein include “Multiple Nucleotide Polymorphisms” (“MNPs”) Insome embodiments, the reference nucleic acid corresponds to a non-drugresistant phenotype and a drug resistant phenotype is detected accordingto a method provided herein by identifying a sequence variation betweenthe reference nucleic acid and a target nucleic acid of a biologicalsample from a subject infected with the microorganism or diseased cell,such as a drug resistant cancer cell.

The terms “responsiveness” and “drug responsiveness” as used herein canrefer to resistance, sensitivity, susceptibility, tolerance and/or otherphenotypic characteristics of a microorganism or diseased cell, such asa cancer cell, related to the therapeutic effect of a drug, includingnon-responsiveness. Drug responsiveness can be assessed directly,according to the effect of the drug on a targeted microorganism ordiseased cell, such as a cancer cell (e.g., a bacterial mortality or acellular mortality), and/or indirectly, according to the effect of thedrug on one or more aspects of an infectious disease caused by themicroorganism (e.g., prevention, amelioration, alleviation, and/orelimination of the disease or one or more symptoms of the disease). Insome preferred aspects, systems and methods are provided herein fordetecting resistance to one or more drugs, where resistance refers toinheritable (genetic) resistance.

The term “variable sequence element” refers to a region of a nucleicacid (e.g., DNA or RNA) comprised of a string of adjacentnucleotides—for example, 2, 3, 5, 10, 15, 25, 50, 75, 100 or moreconsecutive bases—that includes at least one sequence variation known tobe associated with a phenotypic characteristic of interest, such asresistance, sensitivity, and/or other aspects of drug responsiveness.Without being bound by a particular theory, it is believed that sequencevariations associated with drug responsiveness, such as drug resistanceand/or sensitivity, are likely to occur in regions of the nucleic acidthat are important in determining the responsive phenotype, such that avariable sequence element that includes the variation (and surroundingnucleotides) is substantially more likely to contain additional,uncharacterized variations associated with the responsive (e.g.,resistant or sensitive) phenotype. For example, a sequence variationassociated with drug resistance will often occur in a region of anucleic acid that encodes a site of the corresponding protein that is astructural and/or functional determinant of drug responsiveness, such asa drug binding site. A variable sequence element including the knownvariation (and surrounding nucleotides) will likely encode structurallyand/or functionally related portions of the protein (e.g., a pocket,fold, or other structure that comprises the drug binding site), andadditional, uncharacterized variations within the variable sequenceelement will likely be associated with the same phenotype as the knownvariation.

Methods are thus provided herein for assaying drug responsive phenotypesassociated with known and/or unknown sequence variations.Advantageously, such methods are capable of detecting drugresponsiveness without foreknowledge of specific nucleic acid sequencevariations, allowing for rapid identification of new genetic mutationsassociated with drug resistance, drug sensitivity, and/or other drugresponsive phenotypes. As such, methods provided herein can achievegreater sensitivity and diagnostic utility than existing methods basedon characterized mutations.

Accordingly, variable sequence elements are provided herein whichinclude one or more sequence variations known to be associated with adrug resistant phenotype, and assaying such variable sequence elementsas described herein allows detection of the drug resistant phenotype dueto known variations and/or an additional, uncharacterized variation.Advantageously, variable sequence elements provided herein are of a sizethat allows for a high degree of sensitivity together with a low levelof false positives (e.g., a size sufficient to encode the portion of theprotein altered by the known variation(s) and structurally and/orfunctionally related regions without including significant unrelatedportions of the protein). In some embodiments, detection of a sequencevariation within a variable sequence element provided herein isindicative of drug resistance with a false positive rate of less thanabout 25%, less than about 20%, less than about 15%, or more preferablyless than about 10%, 5%, or 1%.

In various aspects, diagnostic methods are provided for determiningwhether a subject infected with a microorganism is amenable to treatmentwith a drug by measuring the responsiveness of the microorganism to thedrug. In some aspects, responsiveness is measured by obtaining abiological sample from a subject, and assaying the sample for one ormore sequence variations within a variable sequence element associatedwith responsiveness to the drug. In some preferred aspects, the variablesequence element is associated with resistance to the drug. In furtherpreferred aspects, the variable sequence element is associated withsensitivity to the drug.

In some preferred aspects, methods are provided for detecting whether asubject is infected with drug-resistant Tb, wherein the method comprisesobtaining a biological sample from the subject and assaying the samplefor one or more nucleic acid sequence variations within a targeted DNAvariable sequence element selected from the variable sequence elementsset forth in Table 1. In some preferred embodiments, methods furthercomprise amplifying targeted variable sequence elements using primersset forth in Table 3.

In some aspects, methods provided herein involve a step of preparing abiological sample to facilitate detection and/or analysis of targetnucleic acids. In some aspects, systems and methods are provided forpreparing a biological sample for high resolution sequence analysis. Insome preferred embodiments, biological samples are treated to amplifytargeted DNA variable sequence elements by polymer chain reaction (PCR),or by other methods known in the art.

PCR amplification generally comprises the steps of initial denaturation,annealing, polymerization, and final extension. PCR amplification isgenerally conducted in a reaction chamber, which is provided withnecessary PCR reagents, including the biological sample containing thetarget DNA, a DNA polymerase (e.g., Taq polymerase), nucleosidetriphosphates, a first and second primer (comprising a primer pair) thathybridize to the target DNA and flank the sequence of the amplified DNAproduct (the “amplicon”). A PCR apparatus will typically include meansfor cycling the temperature of the reaction chamber as required for eachstep of the amplification cycle, including, e.g., “melting” of doublestranded DNA to produce single stranded DNA; annealing of the primers tosingle stranded DNA templates; and extension of the amplified DNA viapolymerase.

The precise conditions used to amplify a specific target DNA sequencecan vary according to a number of factors which are within the knowledgeof skilled artisans. In some embodiments, denaturation is conducted atbetween about 90-95° C. for about 10-30 seconds, annealing is conductedat about 45-65° C. for about 10-30 seconds; extension is conducted atabout 70-75° C. for about 10-90 seconds; and a final extension isconducted at 72° C. for about 5 minutes. In some embodiments, thereaction mixture comprises genomic DNA, MgCl₂ and other physiologicalsalts (e.g., MnCl₂), PCR buffer, 0.1-1.0 mM dNTPs, 0.04-1.5 μM of eachprimer, and 0.5-5.0 units of heat stable polymerase (e.g., Taq.polymerase).

Other amplification methods known in the art may also be utilized,including, for example, self-sustained sequence replication (3SR),strand-displacement amplification (SDA); “branched chain” DNAamplification (Chiron Corp.); ligase chain reaction (LCR), QB replicaseamplification (QBR), ligation activated transcription (LAT), nucleicacid sequence-based amplification (NASBA), repair chain reaction (RCR),and cycling probe reaction (CPR) (reviewed, e.g., in The Genesis Report,DX; Vol. 3(4), pp. 2-7 (February 1994)).

In some aspects, novel primers are provided for use in amplifying targetnucleic acids for analysis according to methods provided herein. Forexample, in various embodiments, the primer pairs set forth in Table 2can be used to amplify the corresponding amplicons set forth in Table 3.which can be used in various methods described herein for detectingsequence variations indicative of drug resistance.

In various aspects, sequence variations are detected within targetnucleic acids according to methods provided herein using melting curveanalysis (MCA). In various embodiments, MCA involves slowly heating DNAfragments in the presence of a dye that allows measurement of therelative amounts of double stranded DNA (dsDNA) and single stranded DNA(ssDNA) as a function of time and temperature, as described, e.g., inMorrison and Stols, Biochemistry, 32: 3095-3104 (1993). Suitable dyesinclude, but are not limited to, dsDNA-specific dyes, such as ethidiumbromide, SYBR Green I, and SYBR Green II (Molecular Probes, Eugene,Oreg.), Eva Green (GENTAUR EUROPE) and ssDNA-specific dyes. In somepreferred embodiments, the dye is a fluorescent dye, such as SYBR GreenI, SYBR Green II, Eva Green, LC Green I, and LC Green Plus. In variousembodiments, dyes can be saturating or non-saturating.

In various aspects, MCA used to detect sequence variations in methodsprovided herein involves incubating a sample containing a target nucleicacid with a nucleotide probe in the presence of a fluorescentDNA-binding dye, and monitoring the degree of hybridization (indicatedby the level of fluorescence) as a function of time and temperature. Forexample, in some embodiments, a variable sequence element from Table 3is amplified in a biological sample, and the amplified sample isincubated with a nucleotide probe complementary to the wild-typesequence set forth in Table 3 in the presence of a dsDNA-binding dye.The sample is then slowly heated at a constant rate (e.g., about 0.05 to10.0° C. per minute) while measuring the level of fluorescence overtime. In various preferred embodiments, a parallel control MCA isconducted, in which the target DNA is known to have the wild-typesequence set forth in Table 3. The target DNA is hybridized to thecomplementary nucleotide probes to form dsDNA at the initial lowtemperatures, while the dsDNA denatures as the temperature increases,converting the dsDNA to ssDNA. The conversion of dsDNA to ssDNA isaccompanied by changes in fluorescence that are characteristic of theparticular dye used. Advantageously, sequence variations in thebiological sample can be detected by analyzing the change influorescence over time relative to that of the control sample.

In various preferred embodiments, MCA used in methods provided hereinallows “high resolution” detection of sequence variations within atarget sequence, which are detected as changes in one or more aspects ofthe fluorescence data. In some preferred aspects, high resolution MCAaccording to methods provided herein can distinguish betweensample-probe and control-probe dsDNA species differing by a single base,and/or by 2, 3, 4, 5, or more bases.

In some aspects, the fluorescence data can be plotted as a function oftime to determine maximum fluorescence, minimum fluorescence, the timeat minimum fluorescence, and a second order rate constant for the knownconcentration of amplified product using the following equation:

$F = {F_{\max} - \frac{F_{\max} - F_{\min}}{{{k( {t - t_{0}} )}\lbrack {D\; N\; A} \rbrack} + 1}}$

wherein F is fluorescence, F_(max) is maximum fluorescence, F_(min) isminimum fluorescence, k is the second order rate constant, t₀ is thetime at F_(min), and [DNA] is the known concentration of the amplifiedproduct. In some embodiments, multiple variables of the fluorescenceversus time data are used to define a group of criteria that serves asan “MCA fingerprint” that uniquely identifies one or more sequencesassociated with a phenotype of interest, such as drug resistance. Forexample, in some embodiments, a drug resistant phenotype can be assayedby conducting MCA using DNA amplified from a biological sample, andcomparing the fluorescence versus time data with an established MCAfingerprint.

In some preferred aspects, methods are provided for assaying abiological sample for drug-resistant tuberculosis, where the methodscomprise amplifying one or more variable sequence elements selected fromTable 3 using one or more of the corresponding primer pairs set forth inTable 2, and assaying the sample for sequence variations within the oneor more amplified variable sequence elements using MCA. In variousembodiments, the detection of one or more variations within a variablesequence element in the biological sample relative to the correspondingvariable sequence element in a control sample or a known standard isindicative of drug resistance.

In various embodiments, amplified MTb DNA comprises one or more of SEQID NOS: 142-145, and a variation in one or more of the amplified DNAsegments indicates sensitivity of MTb to rifampicin; the amplified MTbDNA comprises one or more of SEQ ID NOS: 146-151, and a variation in oneor more of the amplified DNA segments indicates sensitivity of MTb topyrazinamide; the amplified MTb DNA comprises one or more of SEQ ID NOS:152-154, and a variation in one or more of the amplified DNA segmentsindicates sensitivity of MTb to streptomycin; the amplified MTb DNAcomprises one or more of SEQ ID NOS: 155-176, and a variation in one ormore of the amplified DNA segments indicates sensitivity of MTb toisoniazid; the amplified MTb DNA comprises one or more of SEQ ID NOS:177-198, and a variation in one or more of the amplified DNA segmentsindicates sensitivity of MTb to ethambutol; the amplified MTb DNAcomprises one or more of SEQ ID NOS: 199-203, and a variation in one ormore of the amplified DNA segments indicates sensitivity of MTb to oneor both of capreomycin and viomycin; and/or the amplified MTb DNAcomprises SEQ ID NO: 204; and a variation in the amplified DNA segmentindicates sensitivity of MTb to one or more of oxifloxacin,moxifloxican, gatifloxican, sitafloxacin, ofloxacin, levofloxacin, andsparfloxacin.

In some preferred aspects, methods are provided for assaying abiological sample for drug-resistant HIV, where the methods compriseamplifying the variable sequence element of SEQ ID NO: 1 using thecorresponding primer pair of SEQ ID NOS: 1 and 2, and assaying thesample for sequence variations within the amplified sequence using MCA,and wherein the detection of one or more variations within the ampliconof the biological sample relative to a control sample or a knownstandard is indicative of drug resistant HIV. In some preferredembodiments, the detection of one or more variations within the ampliconis indicative of zidovudine and/or nevirapine resistant HIV.

In some preferred aspects, methods are provided for assaying abiological sample for drug-resistant malaria, where the methods compriseamplifying the variable sequence element of SEQ ID NO: 2 using thecorresponding primer pair of SEQ ID NOS: 1 and 2, and assaying thesample for sequence variations within the amplified sequence using MCA,and wherein the detection of one or more variations within the ampliconof the biological sample relative to a control sample or a knownstandard is indicative of drug resistant malaria. In some preferredembodiments, the detection of one or more variations within the ampliconis indicative of chloroquine resistant malaria.

In some preferred aspects, methods are provided for assaying abiological sample for drug-resistant cancer cells, where the methodscomprise amplifying the variable sequence element of SEQ ID NO: 1 usingthe corresponding primer pair of SEQ ID NOS: 1 and 2 and/or the variablesequence element of SEQ ID NO: 2 using the primer pair of SEQ ID NOS: 3and 4, and assaying the sample for sequence variations within one orboth of the amplified sequences using MCA, and wherein the detection ofone or more variations within one or both of the amplicons of thebiological sample relative to a control sample or a known standard isindicative of drug resistant cancer cells. In some preferredembodiments, the detection of one or more variations within theamplicons of SEQ ID NO: 1 and/or SEQ ID NO: 2 is indicative ofepithilone and/or taxane resistant cancer cells.

In some preferred aspects, methods are provided for assaying abiological sample for drug-resistant S. cerivisae, where the methodscomprise amplifying the variable sequence element of SEQ ID NO: 1 usingthe corresponding primer pair of SEQ ID NOS: 1 and 2, and assaying thesample for sequence variations within the amplified sequence using MCA,and wherein the detection of one or more variations within the ampliconof the biological sample relative to a control sample or a knownstandard is indicative of drug resistant S. cerivisiae. In somepreferred embodiments, the detection of one or more variations withinthe amplicon is indicative of terbinafine resistant S. cerivisiae.

In some preferred aspects, methods are provided for assaying abiological sample for drug-resistant S. aureus, where the methodscomprise amplifying the variable sequence element of SEQ ID NO: 1 usingthe corresponding primer pair of SEQ ID NOS: 1 and 2, and assaying thesample for sequence variations within the amplified sequence using MCA,and wherein the detection of one or more variations within the ampliconof the biological sample relative to a control sample or a knownstandard is indicative of drug resistant S. aureus. In some preferredembodiments, the detection of one or more variations within the ampliconis indicative of vancomycin and/or β-lactam resistant S. aureus.

TABLE 1A MTb Nucleic Acid Regions associated with drug resistanceOrganism/Cells Target Region (Gene or region) Drug Resistance/PurposeHIV RT Connector N348I Zidovudine/Nevirapine Malaria ChloroquineResistance Transporter Chloroquine K76T Human cancer cells tubulin BetaT274I epothilone/taxanes Human cancer cells tubulin Beta R282Nepothilone/taxanes S. cerevisiae ERG1 F420L Terbinafine Staphalococcusaureus SA1702 H164R vancomycin/Beta-lactam MTb v176F RNA Polymerase BV176F Rifampicin MTb 80bp HotSpot RNA Polymerase B 80bp hot spotRifampicin MTb CIII a RNA Polymerase B CIIIa Rifampicin MTb CIIIb RNAPolymerase B CIIIb Rifampicin MTb pncA −11 up to codon 105 PyrazinamideMTb pncA codons 254 to 359 Pyrazinamide MTb pncA codons 537 to 545Pyrazinamide MTb pncA codons 128 to 254 Pyrazinamide MTb pncA codons 374to 446 Pyrazinamide MTb pncA codons 464 to 519 Pyrazinamide MTb rpsLcodons 43 to 88 Streptomycin MTb rrs Streptomycin MTb rrs StreptomycinMTb furA detect codon 5 avoid codon Isoniazid 115 MTb ahpC −67 ups tocodon 5 Isoniazid MTb ahpC codon 19 and 32 Isoniazid MTb ahpC codon 73Isoniazid MTb ahpC codon 191 Isoniazid MTb inhA codon 16-95 isoniazidMTb inhA codon 194 isoniazid MTb iniA codon 3 isoniazid MTb iniA codons481 and 537 isoniazid MTb mabA −147 ups to codon 63 isoniazid MTb Rv0340codon 163 isoniazid MTb Rv1592c codon 42 isoniazid MTb Rv1592c codons321 and 322 isoniazid MTb Rv1592c codon 430 isoniazid MTb katG −17 upsto codon 38 isoniazid MTb katG codon 63 to 128 isoniazid MTb katG codons132 to 302 isoniazid MTb katG codons 313 to 350 isoniazid MTb katGcodons 381 and 494 isoniazid MTb katG codons 515 and 595 isoniazid MTbkatG codons 617 and 658 isoniazid MTb katG codon isoniazid MTb embCcodon 394 Ethambutol MTb embC codon 733 Ethambutol MTb embA −43ups tocodon 14 ethambutol MTb embA codon 210 ethambutol MTb embA codons 321and 350 ethambutol MTb embA codon 462 ethambutol MTb embA codons 833 to913 ethambutol MTb embB codons 297 to 332 ethambutol MTb embB codon 406ethambutol MTb embB codon 497 ethambutol MTb embB codon 745 ethambutolMTb embB codons 955 to 1024 ethambutol MTb rmlA2 codon 152 ethambutolMTb iniC codons 245 to 251 ethambutol MTb iniA codon 308 ethambutol MTbiniA codon 501 ethambutol MTb iniB −89ups to codon 47 ethambutol MTbRv3124 −16ups to codon 54 ethambutol MTb RmlD −71ups ethambutol MTb RmlDcodon 284 ethambutol MTb embR −136ups ethambutol MTb embR codon 379ethambutol MTb thyA nt7 to 64 Capreomycin/Viomycin MTb thyA nt 200 to310 Capreomycin/Viomycin MTb thyA nt353 to 400 Capreomycin/Viomycin MTbthyA nt477 to 586 Capreomycin/Viomycin MTb thyA nt 653 to 758Capreomycin/Viomycin MTb gyrA codons 90 and 94 oxifloxacin(Moxifloxacin/ Gatifloxacin/Sitafloxacin/ Ofloxacin/Levofloxacin/Sparfloxacin) MTb—Mycobacterium tuberculosis

The isolation of suitable quantities of Mycobacterium tuberculosis fromsputum samples poses a significant challenge to the molecular diagnosticcommunity. Sputum samples often contain such low quantities of live MTbthat isolates must be grown for up to 2 months to ensure sufficientquantities of genetic material for use in molecular diagnosticapplications. Although many molecular diagnostic techniques can enabledetection of very small quantities of starting materials, as low assingle copy, it is often difficult to ensure that a particular sample infact contains the desired quantity of starting material.

To enable the use of very rare or precious samples in moleculardiagnostic procedures, a technique known as whole genome amplificationhas been employed to enrich the starting material for use in thedownstream molecular diagnostic procedures. As such, some embodiments ofmethods described herein apply whole genome amplification methods to theproblem of screening sputum samples containing MTb. Various methodsprovided herein can also be used for detecting the presence or absenceof one or more nucleic acid sequences in a sample containing a nucleicacid or mixture of nucleic acids, or for distinguishing between twodifferent sequences in such a sample.

In various embodiments, methods are provided for improving the detectionof nucleic acid sequences in biological samples using real-time PCR,dsDNA binding dyes, and fluorescent probe-based approaches.

In some embodiments, methods are provided for preparing microorganismnucleic acids for amplification and high resolution analysis.Microorganisms, such as Mycobacterium tuberculosis (MTb), can beisolated using conventional sample fractionation protocols, and thenucleic acids of the microorganism can be extracted and amplified usingwell-known, novel, or yet to be established methods.

Following molecular enrichment and amplification, the nucleic acids arescreened in various aspects for the presence of any of a variety ofgenetic markers using a quantitative method such as PCR. Nucleic acidsmay also be quantified for downstream analysis. Any method ofquantification may be used, including but not limited to, qPCR analysis,UV analysis, gel analysis, and nucleic acid quantification kits. In someembodiments, a final amplification of nucleic acids is performed toamplify nucleic acids and segments thereof of interest, such as drug(e.g., antibiotic) susceptibility targets. Saturating dyes can be usedto track amplification, hybridization, and denaturation of nucleicacids.

In some embodiments, amplified target nucleic acids can be monitoredduring hybridization and/or denaturation using high resolutionmonitoring techniques, such as those that measure changes influorescence associated with changes in the structure and/orconformation of the nucleic acids, such as those accompanyinghybridization and melting. Control target nucleic acids may be similarlymonitored in parallel. Variations detected between target nucleic acids(e.g., drug susceptibility region) and control nucleic acids can beindicative of reduced susceptibility (e.g., resistance) to drugs thattarget particular regions of the gene products formed from the targetnucleic acids.

In various embodiments, methods are provided for isolating, amplifying,and analyzing target nucleic acids of a microorganism, such as MTb, asdescribed below and throughout the specification. In other embodiments,target nucleic acids, such as those set forth in Table 1, can beisolated from cellular or clinical samples by methods established in theart.

In some embodiments, isolated MTb is fractionated from a sputum sampleusing a conventional live organism preparation method, such as thePetroff method, that leaves small quantities of the MTb organism in anaqueous suspension. MTb nucleic acids are isolated from the sample usinga commercially available kit MycoBuffer, (RAOGene; Milford, Pa.)according to manufacturer's instruction, such that at least smallquantities of MTb DNA are isolated in the residual material from theMycoBuffer product. Larger quantities of DNA may be isolated.

The DNA samples obtained from the use of the lysis solution aresubmitted to a primary screen for MTb DNA using any method of DNAamplification that inhibits or eliminates the formation of nonspecificnucleic acid products. Also, the amplification method can be performedfor extended time periods to account for the low quantity of DNAtypically present in primary lysates. Exemplary primers covering theseregions of interest are presented in Table 2.

TABLE 2 Exemplary primers for amplification of target regions Seq.Organism Target No. Forward Reverse Accession # HIV RT Connector 1AAGGCCAATGGACATATC GGGCACCCCT NP_705927 N348I AAA CATTCTT MalariaChloroquine 3 TATTTATTTAAGTGTATGT CAATTTTGTTT MAL7P1.27 ResistanceGTAATG AAAGTTCTTT Transporter TAGC K76T Human tubulin Beta 5TCCCACGTCTCCATTT TGAGTTCCGG NP_821133 cancer T274I CACTGT cells Humantubulin Beta 5 TCCCACGTCTCCATTT TGAGTTCCGG NM_178014 cancer R282N CACTGTcells S. cerevisiae ERG1 F420L 7 TTCAATGCTAAGAATCCTG AGATTGGCAT M64994CTC ATGATCACTA CC staph SA1702 H164R 9 AAAGCTGCAAATATTAAG GGCAATATAANC_002745 aureus GA CCTGCAC MTb RNA Polymerase 11 GAGCGTGTGGTGGTCAGCGTCTTGTCG BX842579 v176F B V176F GTGGACT MTb 80bp RNA Polymerase 13CAAGGAGTTCTTCGGCACC GGACCTCCAG HotSpot B 80bp hot spot CCCGGCA MTb CIIIa RNA Polymerase 15 GGTGGCACAGGCCAAT GAAGCGACCG B CIIIa TCCGCA MTb CIIIbRNA Polymerase 17 CCGCGCGTGCTGGTC TCCATGTAGT B CIIIb CCACCTCAG MTb pncA−11 to 105 19 CAGTCGCCCGAACGTA TGGTAGTCCG NC_000962 CCGCT MTb pncA 254to 359 21 CAATCGAGGCGGTGTTCT CGACGCCGCG TTG MTb pncA 537 to 545 23GATGCGCACCGCCA GCGGTGCCAT CAGGAG MTb pncA 128 to 254 25 GCGGCGGACTACCATGATTGCCGAC GTGTCCAG MTb pncA 374 to 446 27 GCAACGCGGCGTC CCCTGGTGGCCAAGC MTb pncA 464 to 519 29 GCTTGGCCACCAGGG CTGGCGGTGC GCATC MTb rpsL31 CCGCGTGTACACCACCA AGCGCACACC AF367438 AGGCAG MTb rrs 33GGATTGACGGTAGGTGGA ACGCTCGCAC cp000717.1 GA CCTACGTATTA MTb rrs 35CCCGCCTGGGGAGT CATGCTCCGC L15307.1 CGCTT MTb furA detect 39TAGCCAAAGTCTTGACTG GCGCATTCAC Rv1909c codon 5 avoid AT TGCTTC codon 115MTb ahpC −67 ups to 39 TGTGATATATCACCTTTGC CGGGGAATTG Rv2428 codon 5 CTATCGCC MTb ahpC codon 19 41 ACCAGCTCACCGCTC GGTGATAGTG and 32 GTGAAGTAGTMTb ahpC condon 73 43 GCGTTCAGCAAGCTCA CGCGAATTCG CTGTCA MTb ahpC cond191 45 CTGTGCGCATGCAAC TCCCGGTTAG GCCGA MTb inhA codon 16-95 47CAAACGGATTCTGGTTAG GGTTGATGCC Rv1484 CG CATCCCG MTb inhA codon 194 49CAAGTACGGTGTGCGTT GCCGACGATC Rv1484 GCACTC MTb iniA codon 3 51GAGCCGATTTCACGAACC CTCGTTTACG CCTCAGA MTb iniA codons 481 53TGGGCCGGATGGAATC GACGACGAAC Rv0342 537 GAAATGT MTb mabA −147 ups 55CTGCTGCGCAATTCGTA GATCCCCCGG Rv1483 to codon 63 TTTCCT MTb Rv0340condone 57 GCCGACAGACCATCC GTCGTAGCCG Rv0340 163 TGATGA MTb Rv1592c aa4259 TCCGACGATCCGTTCTAC GAGCGCAACA Rv1592c CCGTTCC MTb Rv1592c aa321 61GACTTCCTCGACGAACC GCCTGCACGA rv1592c 322 TCAATACC MTb Rv1592c aa430 63TTCAACCCGATGACCTACG GGTGATCACC rv1592c TTGGCCG MTb katG −17 ups to 65TGGGGTCTATGTCCTGA GCAGTACCTT Rv1908c codon 38 CAGATTGAG MTb katG codon63 to 67 GGCTCAATCTGAAGGTACT GGGCCAGCTG rv1908c 128 TTAAG MTb katG codon132 69 TTCGCGCCGCTTAAC GGTTCCGGTG rv1908c to 302 CCATAC Mtb katG codon313 71 GTATGGCACCGGAACC TCCTTGGCGG rv1908c to 350 TGTATTG Mtb katG codon381 73 CGCTCCCCGACGATG GACTTGTGGC rv1908c 494 TGCAGG MTb katG codon 51575 CCTGCAGCCACAAGT GCAGGTTCGC rv1908c 595 CTTGTC Mtb katG codon 617 77CGGCCGAGTACATGC GGCTCCCAGG rv1908c 658 TGATAC Mtb katG cddon 79GGCAAGGATGGCAGT GCACGTCGAA rv1908c CCTGT MTb embC394 81 GGCGGGCATGTTTCTGGCGATGATC embC GGCTC MTb embC733 83 GGCGATGATTTCCCAGT GCCAAAGCCT embCGTAGGT MTb embA-4314 85 TCGGCGACAACCTCC GCCCCGGATA embA CCAGAG MTb embA210 87 ACTCGGTTTATCACGACG CCATGGCTAC embA CAGGAC MTb embA321350 89GTATACATCGGTGCTTGC GCACCAGCGG embA TGAACA MTb embA462 FOR 91GCGACCGATGGACTG CCACCACGGT embA GATCAG MMTb embA833913 93CGCCATCACCGACTC TTGCGGTCCG embA ATGTC MMTb embB 297 & 332 95TTCGGCTTCCTGCTCT GGTTTGCTGG embB CCTCC MTb emb 406 97 TCAACAACGGCCTGCATGGACCGCT embB CGATCA MMTb emb 497 99 CACCGTCATCCTGACC TTTTGGCGCG embBAACCC MTb embB 745 101 GGCTGGTCCAACGTG GCATTGGTAT embB CAGGCTCG Mtb embB9551024 103 TTCGCCCGAGCAAAG CCGTTAGTGC embB CGTCT MTb rmlA2 152 105ATGTCACGCTGCAAC GATCCTCCGT rmlA2 CTTCTCCA MTb iniC 245 251 107CGCGAACTGAACCAGA GCGGTATGCG iniC CCTTA MTb iniA 308 109 GAGCAGGTGCTTTCCCCTCTGTTGCC iniA GAACG MTb iniA 501 111 GGGTTCCTATGGCGG GGTTGAACAA iniACCCAAGTC MTb iniB −89 47 113 CGATCCCGATAGGTGTTT GGCACCCAGA iniB TTCAGACMTb Rv3124 −16 54 115 ATCACAGGAGTGGAGTT AAGATGTTGC Rv3124 GCGAAT MTbRmlD −71 117 TACGAACCACACGTTGC GTTGGCTACC RmlD CGACAG MTb RmlD 284 119GCTTGACGCCGCTAC GAAGTTGAGT rmlD TCGCAGGT MTb embR −136 121CAGCCGATGCCGCTG CGCCGATGCG embR GTAAGAA MTb embR 379 123 ACAGCGCCAACGTCAGACGATCGGA embR GGTCGT MTb thyA nt7to64 125 TCGCCGCTAGGCTGA ATCTGCTGGCthyA CGAAC MTb thyA 127 CGGGTACGCCCAAAT CCAGATGGTG thyA ntnt200to310ACTCCG MTb thyA nt353to400 129 ATTCCAATATCGGTTGGC CCACGATCGC thyA CATTGTMTb thyA nt477 to 131 GGTGAGCACATCGACC ATAGCTGGCG thyA 586 ATGTTGA MTbthyA nt 653 to 133 CGCCGACCTGTTTCT CGGCTAGAAG thyA 758 TAGTTTCG MTb gyrA9094 135 GCAACTACCACCCGCA GTAGCGCAGC gyrA GACCA MTb—Mycobacteriumtuberculosis

In further embodiments, DNA samples obtained from the use of the lysissolution are combined, either following the results from the primaryscreen or simultaneous to the screen, with reaction ingredients similarto those used in whole genome amplification procedures.

However, other suitable amplification procedures can be utilized thatenables the DNA samples to be amplified to a suitable amount of genomicnucleic acid. Whole genome amplification procedures can providemolecular enrichment of the DNA samples with increases in quantities ofthe MTb genome in excess of 30 fold in less than 16 hours of incubationtime. Whole genome amplification need only be used if there is notenough template to obtain a primary amplification.

The enriched DNA is subsequently purified using any of a variety ofmethods for purifying DNA. For example, a filter plate system capable ofaccommodating 96 or more simultaneous samples can be used to purify anarray of samples of enriched DNA. The enriched and purified DNA issubjected to a MTb or general mycobacterium-specific PCR amplificationprotocol, and the amount or concentration of the DNA is determined. Forexample, real-time quantitative PCR can be used to amplify and determinethe amount of MTb DNA in the sample. Purification is not necessary ifwhole genome amplification is not used.

The sample concentration is adjusted in order to match the concentrationof the enriched MTb DNA with control MTb DNA so as to achieve a ratio ofapproximately 1:1 or another pre-determined and fixed ratio. This allowsfor a near equivalent ratio of the enriched MTb DNA with that of thecontrol DNA to be used in subsequent detection steps. The enriched MTbDNA that has been normalized for concentration is co-amplified with thecontrol MTb DNA that contains the reference gene sequence for the targetregion of the nucleic acid. That is, the control MTb DNA contains a generegion (e.g., sequence) that if variant, is indicative of a reducedsusceptibility (e.g., resistance) of the MTb organism to drugs (e.g.,antibiotic or antimycotic drugs) targeting the gene region. Exemplarygene regions and corresponding drug sensitivities amplified by theprimer pairs presented in Table 2 are provided in Table 3. These regionsenabled the determination of drug resistance or sensitivity inMycobacterium tuberculosis infection as well as for examples ofZidovudine sensitivity in HIV, taxane sensitivity in human cancers,chloroquine sensitivity in malaria, terbinafine sensitivity in S.cerivisiae, and Vancomycin sensitivity in S. aureus.

TABLE 3 Exemplary regions for drug sensitivity testing of MTb SEQ DesignOrganism Drug Amplicon-Sensitive ID NO Tm Reference HIV Zidovudine/AAGGCCAATGGACAT 137 75.1 www.plosmedicine.org NevirapineATCAAATTTATCAAGA 1890 December 2007 GCCATTTAAAAATCTG volume 4, Issue 12AAAACAGGAAAATAT GCAAGAATGAGGGGT GCCC Malaria ChloroquineTATTTATTTAAGTGTA 138 64.5 The Journal of TGTGTAATGAATAAA InfectiousDiseases ATTTTTGCTAAAAGAA 2001; 183: 1413-6 CTTTAAACAAAATTG Humanepothilone/ TCCCACGTCTCCATTTC 139 84.4 PNAS Mar. 14, 2000 cancer taxanesTTTATGCCTGGCTTTGC vol. 97 no. 6, pages cells CCCTCTCACCAGCCGT 2904-2909GGAAGCCAGCAGTATC GAGCTCTCACAGTGCC GGAACTCA Human epothilone/TCCCACGTCTCCATTTC 139 84.4 PNAS Mar. 14, 2000 cancer taxanesTTTATGCCTGGCTTTGC vol. 97 no. 6, pages cells CCCTCTCACCAGCCGT 2904-2909GGAAGCCAGCAGTATC GAGCTCTCACAGTGCC GGAACTCA S. cerevisiae TerbinafineTTCAATGCTAAGAATC 140 75.3 ANTIMICROBIAL CTGCTCCTATGCACGG AGENTS ANDTCACGTTATTCTTGGTA CHEMOTHERAPY, GTGATCATATGCCAAT December 2003, p.3890-3900 CT Vol. 47, No. 12 S. aureus vancomycin/ AAAGCTGCAAATATT 14171.4 PNAS_May 29, 2007_vol. Beta-lactam AAGGAAAATAATACC 104_no.22_9451-9456 ATTGTTGTTAGACACA TTTTAGGTAAAGTGCA GGTTATATTGCC MTbRifampicin GAGCGTGTGGTGGTC 142 85.7 ANTIMICROBIAL v176F AGCCAGCTGGTGCGGTAGENTS AND CGCCCGGGGTGTACTT CHEMOTHERAPY, CGACGAGACCATTGAC June 2005, p.2200-2209 AAGTCCACCGACAAGA Vol. 49, No. 6 CG MTb 80bp RifampicinCAAGGAGTTCTTCGG 143 90.8 JOURNAL OF HotSpot CACCAGCCAGCTGAGC CLINICALCAATTCATGGACCAGA MICROBIOLOGY, ACAACCCGCTGTCGGG May 2003, p. 2209-2212GTTGACCCACAAGCGC Vol. 41, No. 5 CGACTGTCGGCGCTGG ANTIMICROBIALGGCCCGGCGGTCTGTC AGENTS AND ACGTGAGCGTGCCGGG CHEMOTHERAPY, CTGGAGGTCCOctober 1994, p. 2380-2386 Vol. 38, No. 10 MTb CIIIa RifampicinGGTGGCACAGGCCAA 144 80.9 ANTIMICROBIAL TTCGCCGATCGATGCG AGENTS ANDGACGGTCGCTTC CHEMOTHERAPY, June 2005, p. 2200-2209 Vol. 49, No. 6 MTbRifampicin CCGCGCGTGCTGGTC 145 87.8 ANTIMICROBIAL CIIIb CGCCGCAAGGCGGGCGAGENTS AND AGGTGGAGTACGTGCC CHEMOTHERAPY, CTCGTCTGAGGTGGAC June 2005, p.2200-2209 TACATGGA Vol. 49, No. 6 MTb Pyrazinamide CAGTCGCCCGAACGT 14691.2 ANTIMICROBIAL ATGGTGGACGTATGCG AGENTS AND GGCGTTGATCATCGTCCHEMOTHERAPY, GACGTGCAGAACGACT August 2004, p. 3209-3210TCTGCGAGGGTGGCTC Vol. 48, No. 8; GCTGGCGGTAACCGGT Microbiology (1997),GGCGCCGCGCTGGCCC 143, 3367-3373; GCGCCATCAGCGACTA JOURNAL OFCCTGGCCGAAGCGGCG CLINICAL GACTACCA MICROBIOLOGY, February 2007, p.595-599 Vol. 45, No. 2 MTb Pyrazinamide CAATCGAGGCGGTGT 147 87.9 Asabove TCTACAAGGGTGCCTA CACCGGAGCGTACAGC GGCTTCGAAGGAGTCGACGAGAACGGCACGCC ACTGCTGAATTGGCTG CGGCAACGCGGCGTCG MTb PyrazinamideGATGCGCACCGCCAG 148 83.1 As above GCTCGAGTTGGTTTGC AGCTCCTGATGGCACC GCMTb Pyrazinamide GCGGCGGACTACCAT 149 89.5 As above CACGTCGTGGCAACCAAGGACTTCCACATCGA CCCGGGTGACCACTTC TCCGGCACACCGGACT ATTCCTCGTCGTGGCCACCGCATTGCGTCAGC GGTACTCCCGGCGCGG ACTTCCATCCCAGTCT GGACACGTCGGCAATC MTbPyrazinamide GCAACGCGGCGTCGA 150 88.3 As above TGAGGTCGATGTGGTCGGTATTGCCACCGATC ATTGTGTGCGCCAGAC GGCCGAGGACGCGGTA CGCAATGGCTTGGCCACCAGGG MTb Pyrazinamide GCTTGGCCACCAGGG 151 90.1 As aboveTGCTGGTGGACCTGAC AGCGGGTGTGTCGGCC GATACCACCGTCGCCG CGCTGGAGGAGATGCGCACCGCCAG MTb Streptomycin CCGCGTGTACACCAC 152 91.5 ANTIMICROBiALCACTCCGAAGAAGCCG AGENTS AND AACTCGGCGCTTCGGA CHEMOTHERAPY,AGGTTGCCCGCGTGAA February 1994, p. 228-233 GTTGACGAGTCAGGTC Vol. 38, No.2; GAGGTCACGGCGTACA ANTIMICROBIAL TTCCCGGCGAGGGCCA AGENTS ANDCAACCTGCAGGAGCAC CHEMOTHERAPY, TCGATGGTGCTGGTGC October 2001, p.2877-2884 GCGGCGGCCGGGTGAA Vol. 45, No. 10; GGACCTGCCTGGTGTG JOURNAL OFCGCT BACTERIOLOGY, May 2005, p. 3548-3550 Vol. 187, No. 10; MTbStreptomycin GGATTGACGGTAGGT 153 84.5 As above GGAGAAGAAGCACCGGCCAACTACGTGCCAG CAGCCGCGGTAATACG TAGGGTGCGAGCGT MTb StreptomycinCCCGCCTGGGGAGTA 154 86.7 As above CGGCCGCAAGGCTAAA ACTCAAAGGAATTGACGGGGGCCCGCACAAGC GGCGGAGCATG MTb Isoniazid TAGCCAAAGTCTTGA 155 86.82ANTIMICROBIAL CTGATTCCAGAAAAG AGENTS AND GGAGTCATATTGTCTA CHEMOTHERAPY,GTGTGTCCTCTATACC April 2003, p. 1241-1250 GGACTACGCCGAACAG Vol. 47, No.4; CTCCGGACGGCCGACC TGCGCGTGACCCGACC GCGCGTCGCCGTCCTG GAAGCAGTGAATGCGCMTb Isoniazid TGTGATATATCACCT 156 83 ANTIMICROBIAL TTGCCTGACAGCGACTAGENTS AND TCACGGCACGATGGAA CHEMOTHERAPY, TGTCGCAACCAAATGC March 1997,p. 600-606 ATTGTCCGCTTTGATG Vol. 41, No. 3; ATGAGGAGAGTCATGCANTIMICROBIAL CACTGCTAACCATTGG AGENTS AND CGATCAATTCCCCG CHEMOTHERAPY,August 2006, p. 2640-2649 Vol. 50, No. 8; MTb Isoniazid ACCAGCTCACCGCTC157 85.7 As above TCATCGGCGGTGACCT GTCCAAGGTCGACGCC AAGCAGCCCGGCGACTACTTCACCACTATCACC MTb Isoniazid GCGTTCAGCAAGCTC 158 84 As aboveAATGACGAGTTCGAGG ACCGCGACGCCCAGAT CCTGGGGGTTTCGATT GACAGCGAATTCGCG MTbIsoniazid CTGTGCGCATGCAAC 159 87.4 As above TGGCGCAAGGGCGACCCGACGCTAGACGCTGG CGAACTCCTCAAGGCT TCGGCCTAACCGGGA MTb isoniazidCAAACGGATTCTGGT 160 92.6 ANTIMICROBIAL TAGCGGAATCATCACC AGENTS ANDGACTCGTCGATCGCGT CHEMOTHERAPY, TTCACATCGCACGGGT August 2006, p.2640-2649 AGCCCAGGAGCAGGG Vol. 50, No. 8 CGCCCAGCTGGTGCTCACCGGGTTCGACCGGC TGCGGCTGATTCAGCG CATCACCGACCGGCTG CCGGCAAAGGCCCCGCTGCTCGAACTCGACGT GCAAAACGAGGAGCA CCTGGCCAGCTTGGCC GGCCGGGTGACCGAGGCGATCGGGGCGGGCAA CAAGCTCGACGGGGTG GTGCATTCGATTGGGT TCATGCCGCAGACCGGGATGGGCATCAACC MTb isoniazid AAGTACGGTGTGCGT 161 90.4 As aboveTCGAATCTCGTTGCCG CAGGCCCTATCCGGAC GCTGGCGATGAGTGCG ATCGTCGGCGGTGCGCTCGGCGAGGAGGCCGG CGCCCAGATCCAGCTG CTCGAGGAG MTb isoniazidGAGCCGATTTCACGA 162 84.5 ANTIMICROBIAL ACCGGTGGGGACGTTC AGENTS ANDATGGTCCCCGCCGGTT CHEMOTHERAPY, TGTGCGCATACCGTGA April 2003, p. 1241-1250TCTGAGGCGTAAACGAG Vol. 47, No. 4 Mtb isoniazid TGGGCCGGATGGAAT 163 90.2ANTIMICROBIAL CGAAACCGCTGCGCCG AGENTS AND GGGCCATAAAATGATT CHEMOTHERAPY,ATCGGCATGCGGGGTT April 2003, p. 1241-1250 CCTATGGCGGCGTGGT Vol. 47, No.4 CATGATTGGCATGCTG TCGTCGGTGGTCGGAC TTGGGTTGTTCAACCC GCTATCGGTGGGGGCCGGGTTGATCCTCGGCC GGATGGCATATAAAGA GGACAAACAAAACCG GTTGCTGCGGGTGCGCAGCGAGGCCAAGGCC AATGTGCGGCGCTTCG TCGACGACATTTCGTT CGTCGTC MTb isoniazidCTGCTGCGCAATTCG 164 90.5 ANTIMICROBIAL TAGGGCGTCAATACAC AGENTS ANDCCGCAGCCAGGGCCTC CHEMOTHERAPY, GCTGCCCAGAAAGGGA April 2003, p. 1241-1250TCCGTCATGGTCGAAG Vol. 47, No. 4 TGTGCTGAGTCACACC GACAAACGTCACGAGCGTAACCCCAGTGCGAA AGTTCCCGCCGGAAAT CGCAGCCACGTTACGC TCGTGGACATACCGATTTCGGCCCGGCCGCGG CGAGACGATAGGTTGT CGGGGTGACTGCCACA GCCACTGAAGGGGCCAAACCCCCATTCGTATC CCGTTCAGTCCTGGTT ACCGGAGGAAACCGG GGGATC MTb isoniazidGCCGACAGACCATCC 165 85.3 ANTIMICROBIAL GGCTGTCTGGAACCAC AGENTS ANDCCGGTCGTTGACCCAC CHEMOTHERAPY, ATACCGTCGAGCCCGA April 2003, p. 1241-1250TCATCACGGCTACGAC Vol. 47, No. 4 MTb isoniazid TCCGACGATCCGTTC 166 85.8ANTIMICROBIAL TACTTCCCACCTGCCG AGENTS AND GCTACCAGCATGCCGT CHEMOTHERAPY,GCCCGGAACGGTGTTG April 2003, p. 1241-1250 CGCTC Vol. 47, No. 4 MTbisoniazid GACTTCCTCGACGAA 167 87.4 As above CCCCTTGAGGACATTCTGTCGACGCCGGAAAT TTCCCATGTCTTCGGC GACACCAAGCTGGGTA GCGCGGTGCCCACCCCGCCGGTATTGATCGTG CAGGC Mtb isoniazid TTCAACCCGATGACC 168 84.7 As aboveTACGCCGGCATGGCGA GACTGGCCGTGATCGC GGCCAAGGTGATCACC MTb isoniazidTGGGGTCTATGTCCT 169 88.9 ANTIMICROBIAL GATTGTTCGATATCCG AGENTS ANDACACTTCGCGATCACA CHEMOTHERAPY, TCCGTGATCACAGCCC August 2006, p.2640-2649 GATAACACCAACTCCT Vol. 50, No. 8; GGAAGGAATGCTGTGCANTIMICROBIAL CCGAGCAACACCCACC AGENTS AND CATTACAGAAACCACC CHEMOTHERAPY,ACCGGAGCCGCTAGCA October 2005, p. 4068-4074 ACGGCTGTCCCGTCGT Vol. 49,No. 10; GGGTCATATGAAATAC JOURNAL OF CCCGTCGAGGGCGGCG CLINICALGAAACCAGGACTGGTG MICROBIOLOGY, GCCCAACCGGCTCAAA October 2003, p.4630-4635 GTATACTTTATGGGGC Vol. 41, No. 10 AGCTCCCGCCGCCTTTGGTCCTGACCACCGGG TTGGCCGAGTTTCTGA AGGTACTGC Mtb isoniazidGGCTCAATCTGAAGG 170 94.1 As above TACTGCACCAAAACCC GGCCGTCGCTGACCCGATGGGTGCGGCGTTCG ACTATGCCGCGGAGGT CGCGACCATCGACGTT GACGCCCTGACGCGGGACATCGAGGAAGTGAT GACCACCTCGCAGCCG TGGTGGCCCGCCGACT ACGGCCACTACGGGCCGCTGTTTATCCGGATG GCGTGGCACGCTGCCG GCACCTACCGCATCCA CGACGGCCGCGGCGGCGCCGGGGGCGGCATGC AGCGGTTCGCGCCGCT TAACAGCTGGCCC MTb isoniazidTTCGCGCCGCTTAAC 171 93.6 As above AGCTGGCCCGACAACG CCAGCTTGGACAAGGCGCGCCGGCTGCTGTGG CCGGTCAAGAAGAAGT ACGGCAAGAAGCTCTC ATGGGCGGACCTGATTGTTTTCGCCGGCAACT GCGCGCTGGAATCGAT GGGCTTCAAGACGTTC GGGTTCGGCTTCGGCCGGGTCGACCAGTGGGA GCCCGATGAGGTCTAT TGGGGCAAGGAAGCC ACCTGGCTCGGCGATGAGCGTTACAGCGGTAA GCGGGATCTGGAGAAC CCGCTGGCCGCGGTGC AGATGGGGCTGATCTACGTGAACCCGGAGGGG CCGAACGGCAACCCGG ACCCCATGGCCGCGGC GGTCGACATTCGCGAGACGTTTCGGCGCATGG CCATGAACGACGTCGA AACAGCGGCCCGCCAG CTGTAAGCGCTCTGCAAAGCCGCGTACCGGTA CTTGCTGCAGCTTTGTC GCCGGCTGATCGTCGG CGGTCACACTTTCGGTAAGACCCATGGCGCCG GCCCGGCCGATCTGGT CGGCCCCGAACCCGAG GCTGCTCCGCTGGAGCAGATGGGCTTGGGCTG GAAGAGCTCGTAGCCG GGGCTTGGGCTCCGAC GAGGCGACCTCGTCTACCCGAACCCGACCTTC TCGAGCATTGGCACCG GAACC Mtb isoniazid GTATGGCACCGGAAC172 87.8 CGGTAAGGACGCGATC ACCAGCGGCATCGAGG TCGTATGGACGAACACCCCGACGAAATGGGAC AACAGTTTCCTCGAGA TCCTGTACGGCTACGA GTGGGAGCTGACGAAGAGCCCTGCTGGCGCTT GGCAATACACCGCCAA GGA Mtb isoniazid CGCTCCCCGACGATG 17392.5 As above CTGGCCACTGACCTCT CGCTGCGGGTGGATCC GATCTATGAGCGGATCACGCGTCGCTGGCTGG AACACCCCGAGGAATT GGCCGACGAGTTCGCC AAGGCCTGGTACAAGCTGATCCACCGAGACAT GGGTCCCGTTGCGAGA TACCTTGGGCCGCTGG TCCCCAAGCAGACCCTGCTGTGGCAGGATCCG GTCCCTGCGGTCAGCC ACGACCTCGTCGGCGA AGCCGAGATTGCCAGCCTTAAGAGCCAGATCC GGGCATCGGGATTGAC TGTCTCACAGCTAGTT TCGACCGCATGGGCGGCGGCGTCGTCGTTCCG TGGTAGCGACAAGCGC GGCGGCGCCAACGGTG GTCGCATCCGCCTGCAGCCACAAGTC MTb isoniazid CCTGCAGCCACAAGT 174 91.7 As aboveCGGGTGGGAGGTCAAC GACCCCGACGGGGATC TGCGCAAGGTCATTCG CACCCTGGAAGAGATCCAGGAGTCATTCAACT CCGCGGCGCCGGGGAA CATCAAAGTGTCCTTC GCCGACCTCGTCGTGCTCGGTGGCTGTGCCGC CATAGAGAAAGCAGC AAAGGCGGCTGGCCAC AACATCACGGTGCCCTTCACCCCGGGCCGCAC GGATGCGTCGCAGGAA CAAACCGACGTGGAAT CCTTTGCCGTGCTGGAGCCCAAGGCAGATGGC TTCCGAAACTACCTCG GAAAGGGCAACCCGTT GCCGGCCGAGTACATGCTGCTCGACAAGGCGA ACCTGC Mtb isoniazid CGGCCGAGTACATGC 175 89.4 As aboveTGCTCGACAAGGCGAA CCTGCTTACGCTCAGT GCCCCTGAGATGACGG TGCTGGTAGGTGGCCTGCGCGTCCTCGGCGCA AACTACAAGCGCTTAC CGCTGGGCGTGTTCAC CGAGGCCTCCGAGTCACTGACCAACGACTTCT TCGTGAACCTGCTCGA CATGGGTATCACCTGG GAGCC Mtb isoniazidGGCAAGGATGGCAGT 176 90.3 As above GGCAAGGTGAAGTGG ACCGGCAGCCGCGTGGACCTGGTCTTCGGGTC CAACTCGGAGTTGCGG GCGCTTGTCGAGGTCT ATGGCGCCGATGACGCGCAGCCGAAGTTCGTG CAGGACTTCGTCGCTG CCTGGGACAAGGTGAT GAACCTCGACAGGTTCGACGTGC MTb Ethambutol GGCGGGCATGTTTCT 177 87.6 ANTIMICROBIALGGCTGTCTGGCTGCCG AGENTS AND CTGGACAACGGCCTTC CHEMOTHERAPY,GGCCCGAGCCGATCAT February 2000, p. 326-336 CGCC Vol. 44, No. 2 MTbEthambutol GGCGATGATTTCCCA 178 82.2 ANTIMICROBIAL GTACCCGGCGTGGTCGAGENTS AND GTTGGCCGGTCTAACC CHEMOTHERAPY, TACAGGCTTTGG February 2000, p.326-336 Vol. 44, No. 2 MTb ethambutol TCGGCGACAACCTCC 179 92.9ANTIMICROBIAL GCGGCCCCGCATCCTC AGENTS AND ACCGCCCTTAACCGCG CHEMOTHERAPY,TCGCCTACCATCGAGC February 2000, p. 326-336 CTCGTGCCCCACGACG Vol. 44, No.2 GTAATGAGCGATCTCA CCGGATCGCACGCCTA GCAGCCGTCGTCTCGG GAATCGCGGGTCTGCTGCTGTGCGGCATCGTT CCGCTGCTTCCGGTGA ACCAAACCACCGCGAC CATCTTCTGGCCGCAGGGCAGCACCGCCGACG GCAACATCACCCAGAT CACCGCCCCTCTGGTA TCCGGGGC MTbethambutol ACTCGGTTTATCACG 180 87.5 ANTIMICROBIAL ACGCCCGGCGCGCTCAAGENTS AND AGAAGGCCGTGATGCT CHEMOTHERAPY, CCTCGGCGTGCTGGCG February2000, p. 326-336 GTCCTGGTAGCCATGG Vol. 44, No. 2 MTb ethambutolGTATACATCGGTGCT 181 94.7 ANTIMICROBIAL TGCCCAGCTGGCGGCG AGENTS ANDGTGAGCACCGCCGGCG CHEMOTHERAPY, TCTGGATGCGCCTGCC February 2000, p.326-336 CGCCACCCTGGCCGGA Vol. 44, No. 2 ATCGCCTGCTGGCTGATCGTCAGCCGTTTCGT GCTGCGGCGGCTGGGA CCGGGCCCGGGCGGGC TGGCGTCCAACCGGGTCGCTGTGTTCACCGCT GGTGC MTb ethambutol GCGACCGATGGACTG 182 89.6ANTIMICROBIAL CTGGCGCCGCTGGCGG AGENTS AND TGCTGGCCGCGGCGTT CHEMOTHERAPY,GTCGCTGATCACCGTG February 2000, p. 326-336 GTGG Vol. 44, No. 2 MMTbethambutol CGCCATCACCGACTC 183 94.8 ANTIMICROBIAL CGCGGGCACCGCCGGAAGENTS AND GGGAAGGGCCCGGTCG CHEMOTHERAPY, GGATCAACGGGTCGCA February2000, p. 326-336 CGCGGCGCTGCCGTTC Vol. 44, No. 2 GGATTGGACCCGGCACGTACCCCGGTGATGGG CAGCTACGGGGAGAAC AACCTGGCCGCCACGG CCACCTCGGCCTGGTACCAGTTACCGCCCCGC AGCCCGGACCGGCCGC TGGTGGTGGTTTCCGC GGCCGGCGCCATCTGGTCCTACAAGGAGGACG GCGATTTCATCTACGG CCAGTCCCTGAAACTG CAGTGGGGCGTCACCGGCCCGGACGGCCGCAT CCAGCCACTGGGGCAG GTATTTCCGATCGACA TCGGACCGCAA MMTbethambutol TTCGGCTTCCTGCTC 184 92.9 ANTIMICROBIAL TGGCATGTCATCGGCGAGENTS AND CGAATTCGTCGGACGA CHEMOTHERAPY, CGGCTACATCCTGGGC February2000, p. 326-336 ATGGCCCGAGTCGCCG Vol. 44, No. 2; ACCACGCCGGCTACATGTCCAACTATTTCCGC TGGTTCGGCAGCCCGG AGGATCCCTTCGGCTG GTATTACAACCTGCTGGCGCTGATGACCCATG TCAGCGACGCCAGTCT GTGGATGCGCCTGCCA GACCTGGCCGCCGGGCTAGTGTGCTGGCTGCT GCTGTCGCGTGAGGTG CTGCCCCGCCTCGGGC CGGCGGTGGAGGCCAGCAAACC MTb ethambutol TCAACAACGGCCTGC 185 87.5 ANTIMICROBIALGGCCGGAGGGCATCAT AGENTS AND CGCGCTCGGCTCGCTG CHEMOTHERAPY,GTCACCTATGTGCTGA February 2000, p. 326-336 TCGAGCGGTCCAT Vol. 44, No. 2;MMTb ethambutol CACCGTCATCCTGAC 186 85.4 ANTIMICROBIAL CGTGGTGTTCGCCGACAGENTS AND CAGACCCTGTCAACGG CHEMOTHERAPY, TGTTGGAAGCCACCAG February2000, p. 326-336 GGTTCGCGCCAAAA Vol. 44, No. 2; MTb ethambutolGGCTGGTCCAACGTG 187 87.1 ANTIMICROBIAL CGGGCGTTTGTCGGCG AGENTS ANDGCTGCGGACTGGCCGA CHEMOTHERAPY, CGACGTACTCGTCGAG February 2000, p.326-336 CCTGATACCAATGC Vol. 44, No. 2; Mtb ethambutol TTCGCCCGAGCAAAG188 92.8 ANTIMICROBIAL ATGCCCGCCGATGCCG AGENTS AND TCGCGGTCCGGGTGGTCHEMOTHERAPY, GGCCGAGGATCTGTCG February 2000, p. 326-336CTGACACCGGAGGACT Vol. 44, No. 2; GGATCGCGGTGACCCC GCCGCGGGTACCGGACCTGCGCTCACTGCAGG AATATGTGGGCTCGAC GCAGCCGGTGCTGCTG GACTGGGCGGTCGGTTTGGCCTTCCCGTGCCA GCAGCCGATGCTGCAC GCCAATGGCATCGCCG AAATCCCGAAGTTCCGCATCACACCGGACTAC TCGGCTAAGAAGCTGG ACACCGACACGTGGGA AGACGGCACTAACGG MTbethambutol ATGTCACGCTGCAAC 189 88.9 ANTIMICROBIAL TGGTGCGGGTGGGCGAAGENTS AND CCCGCGGGCATTCGGC CHEMOTHERAPY, TGCGTACCCACCGACG February2000, p. 326-336 AGGAGGACCGCGTAGT Vol. 44, No. 2; CGCCTTTCTGGAGAAGACGGAGGATC MTb ethambutol CGCGAACTGAACCAG 190 93.9 ANTIMICROBIALATGGGCATTTGCCAGG AGENTS AND CGGTGGTGCCGGTATC CHEMOTHERAPY,CGGACTTCTTGCGCTG February 2000, p. 326-336 ACCGCGCGCACACTGC Vol. 44, No.2; GCCAGACCGAGTTCAT CGCGCTGCGCAAGCTG GCCGGTGCCGAGCGCA CCGAGCTCAATAGGGCCCTGCTGAGCGTGGAC CGTTTTGTGCGCCGGG ACAGTCCGCTACCGGT GGACGCGGGCATCCGTGCGCAATTGCTCGAGC GGTTCGGCATGTTCGG CATCCGGATGTCGATT GCCGTGCTGGCGGCCGGCGTGACCGATTCGAC CGGGCTGGCCGCCGAA CTGCTGGAGCGCAGCG GGCTGGTGGCGCTGCGCAATGTGATAGACCAG CAGTTCGCGCAGCGCT CCGACATGCTTAAGGC GCATACCGC MTbethambutol GAGCAGGTGCTTTCC 191 85.5 ANTIMICROBIAL CGCGCGACGGAGCGA AGENTSAND GTGCGTGCTGGGGTAC CHEMOTHERAPY, TCGGCGAAATACGTTC February 2000, p.326-336 GGCAACAGAG Vol. 44, No. 2; MTb ethambutol GGGTTCCTATGGCGG 19282.9 ANTIMICROBIAL CGTGGTCATGATTGGC AGENTS AND ATGCTGTCGTCGGTGGCHEMOTHERAPY, TCGGACTTGGGTTGTT February 2000, p. 326-336 CAACC Vol. 44,No. 2; MTb ethambutol CGATCCCGATAGGTG 193 93.2 ANTIMICROBIALTTTGGCCGGCTTGCGG AGENTS AND ATCAGACCCCGATTTC CHEMOTHERAPY,GGGGTGAGGCGGAATC February 2000, p. 326-336 CATAGCGTCGATGGCA Vol. 44, No.2; CAGCGCCGGTCACGCC GGCGAACAGCTTCTTC GATTGAAGGGAAATGA AGATGACCTCGCTTATCGATTACATCCTGAGC CTGTTCCGCAGCGAAG ACGCCGCCCGGTCGTT CGTTGCCGCTCCGGGACGGGCCATGACCAGTG CCGGGCTGATCGATAT CGCGCCGCACCAAATC TCATCGGTGGCGGCCAATGTGGTGCCGGGTCT GAATCTGGGTGCC MTb ethambutol ATCACAGGAGTGGAG 194 92ANTIMICROBIAL TTTTGAACGCAACGAC AGENTS AND GGCAGGTGCTGTGCAA CHEMOTHERAPY,TTCAACGTCTTAGGAC February 2000, p. 326-336 CACTGGAACTAAACCT Vol. 44, No.2; CCGGGGCACCAAACTG CCATTGGGAACGCCGA AACAACGTGCCGTGCT CGCCATGCTGTTGCTATCCCGGAACCAAGTCG TAGCGGCCGACGCACT GGTCCAGGCAATCTGG GAGAAGTCGCCACCTGCACGAGCCCGACGCAC CGTCCACACGTACATT TGCAACCTTCGCCGGA CCCTGAGCGATGCAGGCGTTGATTCGCGCAAC ATCTT MTb ethambutol TACGAACCACACGTT 195 83.6ANTIMICROBIAL GCGCAGACATCACACT AGENTS AND AGACTACTTGTGTAAC CHEMOTHERAPY,GGCGCCCTGTCGGGTA February 2000, p. 326-336 GCCAA Vol. 44, No. 2; MTbethambutol GCTTGACGCCGCTAC 196 90.5 ANTIMICROBIAL GGCACTGGCGCAGCGCAGENTS AND ACTGGCCACGGCGCTG CHEMOTHERAPY, GCAGCACCTGCGAACT February2000, p. 326-336 CAACTTC Vol. 44, No. 2; MTb ethambutol CAGCCGATGCCGCTG197 90.2 ANTIMICROBIAL TCAAGGGCCACCGACC AGENTS AND CGGTACATCGCACGGCCHEMOTHERAPY, GTGCCGAGATCCTGGG February 2000, p. 326-336TTCTTACCGCATCGGC Vol. 44, No. 2; G MTb ethambutol ACAGCGCCAACGTCA 19888.5 ANTIMICROBIAL GCCGCCACCACGCCGT AGENTS AND CATCGTCGACACGGGCCHEMOTHERAPY, ACCAACTACGTCATCA February 2000, p. 326-336ACGACCTCCGATCGTC Vol. 44, No. 2; MTb Capreomycin/ TCGCCGCTAGGCTGA 19990.3 ANTIMICROBIAL Viomycin CCGCGTGTCAATCGTG AGENTS AND ACGCCATACGAGGACCCHEMOTHERAPY, TGCTGCGCTTCGTGCT August 2005, p. 3192-3197CGAAACGGGTACGCCC Vol. 49, No. 8 AAATCCGACCGCACCG GCACCGGAACCCGCAGCCTGTTCGGCCAGCAG AT MTb Capreomycin/ CGGGTACGCCCAAAT 200 88.8ANTIMICROBIAL Viomycin CCGACCGCACCGGCAC AGENTS AND CGGAACCCGCAGCCTGCHEMOTHERAPY, TTCGGCCAGCAGATGC August 2005, p. 3192-3197GCTATGATTTGTCGGC Vol. 49, No. 8 CGGTTTCCCGCTGCTC ACTACCAAGAAAGTCCATTTCAAATCGGTAGC CTACGAGCTGCTGTGG TTTTTGCGCGGCGATT CCAATATCGGTTGGCTGCACGAGCACGGAGTC ACCATCTGG MTb Capreomycin/ ATTCCAATATCGGTT 201 84.7ANTIMICROBIAL Viomycin GGCTGCACGAGCACG AGENTS AND GAGTCACCATCTGGGACHEMOTHERAPY, CGAATGGGCAAGTGAT August 2005, p. 3192-3197ACAGGCGAACTCGGGC Vol. 49, No. 8 CGATCTACGGTGTACA ATGGCGATCGTGG MTbCapreomycin/ GGTGAGCACATCGAC 202 90.9 ANTIMICROBIAL ViomycinCAGATCAGCGCGGCGC AGENTS AND TGGATTTGCTGCGCAC CHEMOTHERAPY,CGATCCCGATTCCCGG August 2005, p. 3192-3197 CGCATCATCGTGTCGG Vol. 49, No.8 CCTGGAACGTCGGCGA AATCGAGCGGATGGCG CTGCCGCCCTGTCATG CGTTCTTCCAGTTCTACGTCGCCGATGGCCGGC TGAGCTGTCAGCTCTA CCAACGCAGCGCCGAC CTGTTTCTGGGTGTGCCGTTCAACATCGCCAG CTAT MTb Capreomycin/ CGCCGACCTGTTTCT 203 89.9ANTIMICROBIAL Viomycin GGGTGTGCCGTTCAA AGENTS AND CATCGCCAGCTATGCCHEMOTHERAPY, GTTGCTCACCCACAT August 2005, p. 3192-3197 GATGGCCGCCCAGGCVol. 49, No. 8 CGGCTTGTCGGTCGG CGAGTTCATCTGGAC CGGTGGCGACTGCCACATCTACGACAATCA CGTCGAGCAAGTACG GCTGCAGCTCAGCCG CGAGCCGCGGCCATATCCGAAACTACTTCT AGCCG MTb oxifloxacin GCAACTACCACCCGC 204 88.3ANTIMICROBIAL Moxifloxacin/ ACGGCGACGCGTCGAT AGENTS AND Gatifloxacin/CTACGACAGCCTGGTG CHEMOTHERAPY, Sitafloxacin/ CGCATGGCCCAGCCCT August2005, p. 3192-3197 Ofloxacin/Levofloxacin/ GGTCGCTGCGCTAC Vol. 49, No. 8Sparfloxacin) MTb—Mycobacterium tuberculosis

The co-amplified sequences of enriched MTb DNA and control MTb DNA aresimultaneously denatured, and then annealed to produce homoduplexes ofamplified control MTb DNA and enriched MTb DNA, and also produceheteroduplexes of the control and enriched MTb DNAs. A saturatingdouble-stranded DNA binding dye, such as a dye that fluoresces wheninteracting with a duplexed nucleic acid, is included in theamplification mixture to enable the generation of high resolutionmelting curve data from these homoduplexes and heteroduplexes. As such,the annealed samples of homoduplexes and heteroduplexes as well as thecontrol MTb DNA are subjected to high resolution melting curve analysisthat is monitored using fluorescence or other methods of detecting thebinding dye.

The data obtained from monitoring the high resolution melt of thehomoduplexes, heteroduplexes, and control MTb DNA are input into acomputing system to analyze the data. A mathematical comparison of thecontrol MTb DNA sample data without added enriched sample DNA is thencomputed against the sample containing the co-amplified homoduplexes andheteroduplexes. The mathematical comparison, after normalization of thecurves by temperature and beginning and ending points, allows thesubtraction of each data point along the melting curve of the samplecontaining the co-amplified product from the control MTb DNA sampledata. The resulting graph for invariant samples that have sequences thatare not substantially different from the control MTb DNA is essentiallya flat line with minor variation about zero. A graph for samples thathave heteroduplex DNA (e.g., control DNA with enriched sample DNA) thatcontains base pairing mismatches will show a change in the meltingcurve, and when subjected to the subtraction algorithm will produce adistinctly different graph than the flat graph of control and invariantsequences.

Samples that contain variant graphs from the control sample graphs arescored as variant in the drug target region (e.g., nucleic acid target),and microorganisms are likely to be less susceptible (e.g., resistant)to the action of the drug for this genetic region. Also, several drugtarget nucleic acid regions can be amplified simultaneously in differentreaction chambers for a single patient or for multiple patients.

In various aspects, the systems and methods enable rapid screening forsuitable drugs for the treatment of individual cases of MTb. Using suchan approach, a rapid personalized pharmaceutical regimen can beprescribed to a MTb patient, which can result in fewer drugs perpatient, higher rates of compliance to treatment regimens, and/or anultimate reduction in the rate of MDR-MTb generation.

II. Novel Primers

In some embodiments, methods are provided for improving the detection ofnucleic acid sequences by utilizing rational oligonucleotide primerdesigns and rational target sequence designs in combination to producenarrow temperature ranges for both the annealing of primers with thetarget nucleic acid, amplification of the target nucleic acid, anddenaturation of the amplified target nucleic acid product. As such,narrowed temperature ranges compared to the temperature range normallyemployed can result in an amplified target nucleic acid product thatcontains fewer nonspecific products. Thus, the amplified target nucleicacids products can be overall more specific and sensitive forquantitative PCR and genotyping target detection applications asdescribed herein.

Rational design of oligonucleotide primers can include the selection viacalculation, experiment, or computation of primers that have the desiredmelting temperature (Tm). The rational design can include selection of aspecific primer sequences with the appropriate CG % to obtain thedesired Tm. Also, the rational design can include modifications to theprimers that include internucleotide modifications, base modifications,and nucleotide modifications.

In some embodiments, methods are provided for selecting primers for PCRthat flank a variable sequence element of interest on a target nucleicacid. In some embodiments, primers are selected to have a Tm with thetarget nucleic acid (primer:target Tm) that is within a narrow range ofthe Tm of the target nucleic acid (target:target Tm). The specific,narrow temperature range used for such an amplification of the targetnucleic acids is dependent on the melting profile of the target nucleicacid, and thereby the sequence of the target nucleic acid beingamplified. As such, the narrow temperature range can be used as a targettemperature range in order to identify and/or generate specific primersthat have sufficiently high Tm values when hybridized with the targetnucleic acid. Accordingly, the Tm values of the primers can beoverlapping within the temperature range of annealing and/or denaturingof the target nucleic acid (see, FIG. 1).

FIG. 1 can be contrasted with FIG. 2 to illustrate the design of theprimers to have the Tm within a range of the Tm of the target nucleicacid. FIG. 2 shows that conventional amplification with primers and atarget nucleic acid are devoid of having a temperature overlap (as shownin FIG. 1) and require extreme temperature variations duringamplification, corresponding to denaturation, annealing and extensioncycles, to produce an amplified product. Such extreme temperature rangesallow for the formation of undesired products.

In some embodiments, an iterative design process is provided to selectand/or optimize primers for specific target nucleic acid sequences to beamplified and/or detected. Advantageously, the iterative method enablesthe formation of a specific target nucleic acid by using a narrow rangeof thermal conditions where both the target nucleic acid and theoligonucleotide primers hybridized with the target nucleic acid are in adynamic flux of annealing and denaturing. Such a dynamic flux ofannealing and denaturing can result in a specific amplification of thetarget nucleic acid with a commensurate decrease in the formation ofnonspecific amplification products.

The implications of such iterative methods for selecting and/oroptimizing primers provides for the use of low cost dyes in lieu of moreexpensive custom oligonucleotide probes, such as those havingfluorescent labels, can allow for quantitative PCR or high resolutiondenaturation to be used in analyzing the sequence of the target nucleicacid. Also, the iterative method can provide primers that function inthe absence of exquisite thermally-controlled instruments for theformation of amplification products. That is, the primers can operatewithin a narrow temperature range in order to amplify the target nucleicacid, allowing nucleic acid amplification to be used in a much broaderrange of uses.

A number methods have been described in the art for calculating thetheoretical Tm of DNA of known sequence, including, e.g., methodsdescribed by Rychlik and Rhoads, Nucleic Acids Res. 17:8543-8551 (1989);Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); and Breslaueret al., Proc Natl Acad. Sci. 83: 3746-3750 (1986).

In some embodiments, primers can be configured to have a Tm with thetarget nucleic acid that is within a narrow range of the Tm of thetarget nucleic acid by chemically modifying the oligonucleotides. Wellknown oligonucleotide synthesis chemistries may be used to increase theTm values of the primers so they correspond to the temperature range ofthe Tm of the target nucleic acid. Such chemistries may use modifiedbases (e.g., Super G, A, T, C), LNA, or PNA, or other sucholigonucleotide stabilizing chemistries. Also, additionaloligonucleotide hybridization stabilizing chemistries may be developedthat can be used for this application.

For example, primers synthesized with both conventional phosphodiesterlinkage chemistry, and LNA chemistries have been used to provide primerTm values close to the Tm values of the target nucleic acid sequence.However, it is possible that certain target nucleic acids may have Tmvalues lower than that of the primers, and a hybridization destabilizingchemistry may need to be included to decrease the primer Tm values sothat the primer Tm value is within a range of the Tm values of thetarget nucleic acid sequence.

In some embodiments, methods are provided for refining the design of theprimers to minimize the temperature range for the specific amplificationof the target nucleic acid sequence. As such, the target nucleic acid isamplified with standard reaction thermal cycling conditions to ensurethe target nucleic acid sequence is amplified. The amplification ismonitored using real-time PCR with a double-stranded DNA binding dye,such as SYBR, LCGreen, LCGreen+, Eva dye, or the like. The amplifiedtarget nucleic acid is subjected to a melting curve analysis todetermine the actual Tm value of the target nucleic acid sequence. Themelting peak, which can be expressed as −dF/dT, is generated frommelting the amplified target nucleic acid and can have a range similarto a distribution curve across a defined temperature range. At the lowtemperature end, the amplified target nucleic acid template is partiallydenatured. At the uppermost temperature the entire sample of amplifiedtarget nucleic acid is denatured. The temperature necessary to denaturethe target nucleic acid during the amplification procedure is withinthis temperature distribution. Initially, the uppermost temperature isrecommended to ensure more complete denaturation. Subsequently, thelowermost temperature of the distribution curve can be used as theinitial Tm for a set of designed primers for use in amplification beforeany iterative changes are made to the primers. Confirmation of thenarrow temperature range that the initial primers may be used with canbe performed either in serial or in parallel experiments of everincreasing annealing temperatures. Alternatively, the individual primerscan be added to the amplified template and an additional melting curveanalysis can be performed on the combined primer and template meltingcurves. In any event, the Tm of the primers can be configured to overlapwith a narrow temperature range that contains the Tm of the targetnucleic acid sequence.

The highest annealing temperature from these experiments where thetarget nucleic acid sequence is amplified specifically and efficientlycan be considered the temperature which defines the optimal annealingtemperature for the existing primers (e.g., primers that were tested).These same primers or slightly modified primers can then bere-synthesized with additional hybridization stabilizing chemistries.Modifications to the primers to change the Tm in the desired directionso that the primer Tm overlaps with a narrow temperature range thatcontains the Tm of the target nucleic acid sequence. This can beaccomplished using online design tools, such as the LNA design toolavailable from Integrated DNA Technologies. Such design tools can beused to estimate the number of necessary LNA modifications required toraise the Tm of the primer to better overlap with the melting curve ofthe target nucleic acid sequence.

In the instance the primer Tm values are greater than the highestmelting temperature of the target nucleic acid sequence, it may benecessary to redesign the primers to have a lower Tm. Alternatively, thequantity of divalent and/or monovalent cation salts or otherdestablizing agents (e.g., AgCl, DMSO, etc.) that are used in theamplification protocol (e.g., PCR) may be reduced to destabilize thehybridization of these oligonucleotides to the template. In any event, areduction in the primer Tm may be needed in some instances.

In some embodiments, the primers can be prepared so that the targetnucleic acid amplification or enrichment protocols can be performed atminimized temperature differences during the thermal cycling. Thisallows the thermal cycling to be done within a narrow temperature rangeso as to promote the formation of a specific product. One range ofthermal cycling can be within about 15° C. of the target nucleic acidTm, more preferably within 10° C., even more preferably within 5° C.,still more preferably within 2.5° C., and most preferably substantiallythe same Tm as that of the target nucleic acid Tm. For example, thethermal cycling conditions for the amplification of the target nucleicacid spans the range of the Tm peak+/− about 5 to 10° C. of the targetnucleic acid sequence. Such narrow temperature ranges make it possibleto amplify specific target nucleic acids without thermal cycling betweentemperatures corresponding to the normal stages of PCR amplification(denaturation, annealing and extension). Also, it makes it possible toperform amplifications and enrichments in commercialtemperature-controlled instruments that can be set at selectedtemperatures or be varied within narrow temperature ranges, such as anoven, heating block, or the like. FIG. 3 illustrates the graph of anarrow temperature range PCR amplification with the same target nucleicacid sequence as shown in FIG. 2, which shows more specific productformation and less undesired products are formed.

In some embodiments, the temperatures of the thermocycling can beselected in a narrow temperature range to substantially limitamplification to amplifying the target nucleic acid sequence. As such,the thermal cycling conditions can be modified to amplify the targetnucleic acid sequence by modifying the annealing temperature to besubstantially the same as the lower temperature base of the melting peakfor the amplicon. Also, the thermal cycling conditions can be modifiedto amplify the target nucleic acid sequence by modifying the annealingtemperature to be substantially the same as the higher temperature basefor the melting peak of the amplicon.

In some embodiments, the primer Tm can be selected so that theamplification of the target nucleic acid can be performed at atemperature that ranges between about 75 to about 90° C. Such atemperature range, or narrowed 5 to 10° C. range therein, can be usedfor the amplification of DNA and/or RNA target nucleic acid sequences toreduce the formation of non-specific products during the amplification(e.g., PCR) process.

In some embodiments, the primer Tm can be selected so that theamplification is performed at isothermal amplification conditions in theTm range of the target nucleic acid sequence to ensure appropriateproduct formation.

In some embodiments, the present invention includes a method ofdesigning a primer set having a Tm with a target nucleic acid that iswithin a narrow range from the Tm of the target nucleic acid sequence.As such, the primer set can be designed so that the primer Tm overlapsthe distribution curve of the Tm of the target nucleic acid sequence.For example, the primer set can be used in real-time PCR assays so thatthe primer Tm overlaps the distribution curve of the Tm for the targetnucleic acid sequence so that a narrow temperature range can be used toamplify the target nucleic acid sequence. For example, the primer can bedesigned so as to have a primer Tm that is within about 15° C. of thetarget nucleic acid Tm, more preferably within 10° C., even morepreferably within 5° C., still more preferably within 2.5° C., and mostpreferably substantially the same Tm as that of the target nucleic acidTm. Also, this can include primer Tm values that overlap with theamplicon Tm curve.

In some embodiments, the present invention includes an iterative processfor designing primers. Such an iterative process can include identifyingan initial target nucleic acid sequence as the target amplicon, whereinthe target nucleic acid sequence can be associated with a particularbiological activity, such as possible drug resistance. The targetnucleic acid sequence is then amplified in order to produce an amplifiedproduct, and the Tm value of the amplified product (e.g., amplicon) isdetermined using conventional melting curve analysis. The melting curveanalysis is then utilized to determine or compute new primers or primersets for use in the amplification of the target nucleic acid. Thedetermined or computed primers are then designed with primer Tm valueswithin the range of the melting peak generated by the melt of theamplified product. The primers are then prepared or synthesized to havethe designed primer Tm values.

In some embodiments, the conditions of the protocol for amplifying thetarget nucleic acid sequence can be modified to an appropriate pH toincrease the specificity of selectively amplifying the target nucleicacid over other nucleic acids. As such, the use of an appropriate pH canincrease the ability to selectively amplify the target nucleic acidsequence. This can include the use of an amplification buffer that canenable the activation of chemically inactivated thermal stable DNApolymerases. Also, adjusting the pH with selected amplification bufferscan allow for the amplification protocol to be performed at reducedtemperatures, such as those temperatures ranges that have been recitedherein.

In some embodiments, the pH of the amplification buffer can be adjustedso as to allow for the conversion of a chemically inactivated enzyme tothe activated state. As such, an enzyme may be activated in a slightlyacidic condition; however, basic pH values may be used for some enzymes.For acid-activated enzymes, standard Tris-based PCR buffers can havesignificant temperature dependence (e.g., reducing by 0.028 pH units perdegree C.). Complete activation of the enzyme (e.g., chemicallyinactivated thermal stable DNA polymerases) from the inactivated stateof can require the pH to be less than about 7, more preferably less thanabout 6.75, and most preferably less than 6.5.

In some embodiments, the amplification protocol includes the use oflower pH buffers so that the amplification can be performed at loweractivation temperatures. For example, for every 10° C. below 95° C., theenzyme activation temperature can be lowered by 0.3 pH units. However,limits to this approach are entirely a function of the dye chemistryused for the real-time detection of the amplified template (e.g.,Fluorescein-based detection has significantly reduced fluorescence belowpH 7.3).

In some embodiments, the primer Tm can be modified by altering the GC %of the primer sequence. By changing the GC %, the primer Tm can beselectively changed. Usually, increasing the GC % can increase the Tm,and decreasing the GC % can decrease the Tm. However, there areinstances that a high GC % is desired that will overly increase the Tm.In such instances, destabilizers can be used to enable the inclusion ofhigh GC % content primers or for the use of high GC % target nucleicacid sequences. The de-stabilizers can selectively decrease thetemperature of the amplification procedure. Examples of destabilizersinclude DMSO, AgCl, and others.

In some embodiments, the design of the primers and/or amplificationconditions can be modulated so as to modulate the size of the targetnucleic acid sequence being amplified. This can include modulating thedesign of the primers and/or amplification conditions so that the sizeof the amplicon is significantly larger than that of the combinedprimers only. This can include the amplicon being 1-3 nucleotides longerthan the primers, or 2 times larger than the primers, or 5 times largerthan the primers, and more preferably 10 times larger than the primers.

In some embodiments, the primers designed as described herein can beemployed in an array of amplification procedures with differentconcentrations of starting material. That is, the starting material canbe partitioned into an array at varying concentrations, and the primerscan be used therewith for the narrow temperature amplification protocolas described herein. The use of the primers and narrow temperatureamplification protocol with an array of varying concentrations ofstarting material can be used for quantification of the amount of targetnucleic acid in the starting material. FIG. 4 is a graph that shows theuse of the primers and protocol with an array of varying concentrationsof starting material so that the amount of target material can bequantified.

III. Target Nucleic Acid Amplification/Enrichment

In some embodiments, methods provided herein include a step ofamplifying or enriching the target nucleic acid. Such a method caninclude a procedure substantially similar to well-known methods of wholegenome amplification and whole transcriptome amplification. This caninclude amplifying a genome with a genome library generation step, whichcan be followed by a library amplification step. Also, the librarygenerating step can utilize the specific primers or mixtures of thespecific primers described herein with a DNA polymerase or ReverseTranscriptase. The specific primer mixtures can be designed with theprimers so as to eliminate ability to self-hybridize and/or hybridize toother primers within a mixture, but allow the primers to efficiently andfrequently prime the target nucleic acid sequence, wherein the primerscan be designed as described herein.

In some embodiments, methods are provided for simultaneously determininga genetic expression profile for an individual member of a speciesrelative to an entire standard genome for the species. The methods cancomprise distributing a liquid sample of genomic material into an arrayof reaction chambers of a substrate. The array can comprise a primer setand a probe for each target nucleic acid sequence along the entirestandard genome. The liquid sample can comprise substantially allgenetic material of the member. Each of the reaction chambers cancomprise the primer set and the probe for at least one of the targetnucleic acid sequences and a polymerase. The methods can furthercomprise amplifying the liquid sample in the array, detecting a signalemitted by at least one of the probes, and identifying the geneticexpression profile in response to the signal.

Since the isolation of suitable quantities of microorganisms, such asMTb, from sputum samples can be a significant challenge, the genomeamplification techniques described herein can be used instead oftraditional culturing and purification protocols. Although manymolecular diagnostic techniques enable the detection of very smallquantities of starting genetic material (e.g., as low as a single copyof a target nucleic acid sequence), it is often difficult to ensure thata particular sample actually contains the desired single copy of thetarget nucleic acid sequence. To enable very rare or precious samples tobe tested accurately in molecular diagnostic procedures, a techniqueknown as whole genome amplification has been employed to enrich thestarting material for use in the downstream molecular diagnosticprocedures. The method described here applies the whole genomeamplification method to the problem of MTb screening of sputum sampleswhich often contain such low quantities of live organism. Otherwise,standard procedures may use isolates of MTb that must be grown for up to2 months to ensure sufficient quantities of genetic material can beobtained from the sample for molecular diagnostic applications.

Using whole genome amplification techniques developed for the in vitroenrichment of rare and precious DNA and/or RNA samples, a novel geneticmaterial enrichment method has been developed to enrich samplescontaining a microorganism DNA, such as MTb DNA. This technique enablesthe circumvention of conventional culturing methods that have heretoforebeen used to increase concentrations of microorganisms, which are oftenrequired for downstream molecular diagnostics. Such a whole genomeamplification technique uses small quantities of genomic DNA fromdirectly lysed microorganism samples. Samples containing livemicroorganism that have been isolated using the Petroff method can bedirectly lysed by a commercially available product, and the resultingsmall quantities of microorganism DNA can be subjected to the wholegenome amplification techniques to provide an amplicon for use indownstream molecular diagnostic applications. While the procedure foremploying the whole genome amplification technique is described withrespect to MTb, it is recognized that such a technique can be applied toany microorganism.

Using a conventional live organism preparation method, the Petroffmethod, the isolated MTb is fractionated from the sputum sample leavingsmall quantities of the organism in a suspension of water. Following theprotocol of the manufacturer of the mycobacterium lysis solution,MycoBuffer, (RAOGene; Milford, Pa.), small quantities of MTb DNA areisolated in the residual material from the MycoBuffer product. Usingthis directly lysed DNA sample and combining it with reactioningredients similar to those used in whole genome amplificationprocedures enables molecular enrichment of the sample DNA. Such aprocedure can provide increased quantities of the MTb genome, forexample, in excess of 30 fold in less than 16 hours of incubation time.This level of sample enrichment can produce sufficient quantities of MTbgenomic material to enable the use of this enriched material indownstream molecular diagnostic procedures in less than a day comparedto current methods that may take more than 2 months of MTb culturing ofthe MTb isolates prior to diagnostic testing.

The whole genome amplification technique may be used with one or manyDNA polymerases in order to improve the enrichment results either byreducing the time required for enrichment or by increasing the quantityof resultant enriched material. This can be used for amplifying RNAand/or DNA. Also, the amplification technique may be used with reversetranscriptase enzymes either alone or in combination with DNA polymeraseenzymes to enrich samples for RNA components of the lysed material.Additionally, the amplification technique may be used with one or manydifferent target nucleic acid priming parameters. Examples of thepriming parameters that can be modulated include the following: the sizeprimers; random primers; quantity of random primers; specific targetprimers; region specific primers; and combinations thereof. Modulationof such priming parameters can improve the whole genome amplification orspecific region amplification within the samples. Further, theamplification technique may be used with various buffer mixes to improvethe enrichment of the sample. Furthermore, the amplification techniquemay be used with various concentrations of nucleic acid building blocks,which may come from natural or synthetic sources. Further still, theamplification technique may be performed in any instrument capable ofmaintaining a constant temperature or varying temperature through anarrow temperature range (e.g., an instrument capable of maintaining aset temperature, either stably or with programmable thermal profiles).The reaction conditions can include some temperature variation withinthe temperature range during the enrichment process in order to improvethe quantity of enriched genetic material or to specify the enrichmentof specific regions of the genetic material, such as the target nucleicacid sequence.

For example, the sample genomic material may be isolated using anymethod that will release the microorganism (e.g., MTb) nucleic acidsinto solution or into a solid phase collector. The sample genomicmaterial may be isolated from samples other than sputum, such as, butnot limited to, blood, cerebral spinal fluid, skin lesions, organlesions, or from environmental samples. The sample genomic material maybe enriched using an enrichment method similar to whole genomeamplification or nested PCR amplification. This can allow for regionssurrounding the target nucleic acid sequence to be amplified using athermal cycling method in combination with specific primers (e.g.,primers having a Tm as described herein) to amplify the target nucleicacid sequence. Also, non-specific primers may be used to amplify thegenome in a type of genome wide nested PCR.

Mycobacterium tuberculosis nucleic acid sample in the Mycobuffersolution can be prepared from the nucleic acid extraction protocolprovided by the vendor or by any standard method. The nucleic acid maybe either DNA or RNA from the microorganism sample to be enriched, wherethe nucleic acid can be intact, fragmented, or portions of the entireorganisms nucleic acid. The enrichment mixture can include suitable DNAand/or RNA polymerase buffers, deoxynucleotide triphosphates, saltsappropriate for the specific enzyme and buffer system, and randomoligonucleotide primers. Examples of primer length can include 6 base,11 base, and 22 base primers. The primers can be phosphodiesteroligonucleotides, LNA oligonucleotides, PNA oligonucleotides, or anycombination of thereof; however, future chemistries that can produceamplification or an enrichment of the interrogated target DNA or RNA arealso expected to function properly in this technique. Also included inthe mix may be a single-stranded DNA or RNA binding protein to improvethe overall performance of the enrichment step.

An exemplary amplification technique can be performed as follows: thetest sample target nucleic acids are combined in a suitablepolymerization buffer with appropriate salts, with a randomoligonucleotide primer (e.g., 6, 11, or 22 bases, or any of the primersor lengths of primers presented in Table 2), and the nucleic acids aredenatured at a temperature high enough to ensure that denaturation is atleast substantially complete, preferably complete; the denatured samplesare maintained at near denaturing conditions, or in a temperatureenvironment that will enable the target nucleic acid sequence of thesample to experience destabilized hybridization conditions; the samplesare then cooled sufficiently to allow the primers to anneal to thetarget sequence, wherein the target sequence is contained within eitherthe whole genome or fragments thereof; appropriate nucleic acid buildingblocks are added to the mix, which are either deoxynucleotidetriphosphates, or ribonucleotide triphosphates, or possibly unnatural orartificial nucleic acid bases which can be incorporated with theproducts formed; appropriate enzymes (e.g., DNA polymerase, RNApolymerase, reverse transcriptase, any combination thereof, or the like)for the enrichment objectives are then combined; and the amplificationis conducted at the narrow temperature in order to selectively amplifythe target nucleic acid sequence.

IV. Screening Target Nucleic Acid to Determine Drug Resistance

In some embodiments, the amplified nucleic acids described herein can beemployed in a method for screening the target nucleic acid sequence forthe presence and/or absence of nucleic acid sequences or change innucleic acid sequence indicative of drug resistance. That is, theamplified nucleic acids can be screened for a selected nucleic acidsequence by using high resolution denaturation in order to determinewhether the microorganism may be drug resistant to a selected drug. Assuch, molecular diagnostics of nucleic acids can be used to detectgenetic changes in target nucleic acid sequences, where changes in thesequence can be an indication that the microorganism is resistant to adrug. Accordingly, known genetic sequences that are altered in drugresistant strains can be analyzed to determine whether there are anysuch alterations in the gene sequences. Such genetic alterations areoften indicative of altered susceptibility of the pathogen to treatmentby drugs, which is often manifest by being drug resistant.

Typically, existing techniques require a foreknowledge of the specificmutations in the genetic nucleic acids that are related to drug targets(e.g., the nucleic acid is either the drug target or produces a geneproduct that is the drug target). This information is used to screen fordrug resistance, and any changes in the pathogen's genetic material thatis not being tested for specifically may be overlooked during thescreening process. The method described herein does not require anyforeknowledge of the specific changes. As such, a general region of thepathogen's genetic nucleic acids (e.g., DNA, RNA, etc.) is studied tosee whether there are any variations in the sequence that is either thedirect target of the drug or encodes for the direct target of the drug.Also, changes in the genetic material in such a region of the pathogen'sgenetic nucleic acids may render individual therapeutic drugsineffective or reduce their efficacy. This technique allows the rapididentification of any genetic changes to drug target nucleic acidsequences, and can provide greater sensitivity in being capable ofdetecting expected as well as unexpected changes in the drug targetnucleic acid sequences. Accordingly, the methods of the presentinvention can be used to generate drug sensitivity profiles of anyparticular microorganism isolate so that the likelihood of drugresistance can be established.

In some embodiments, the method of screening includes determiningwhether a specific microorganism is present in a sample. Also, theamount of the microorganism genetic material can be determined. Anypositive samples are then processed herein in order to amplify theamount of genetic material. This can include combining the sample withprimers or primer sets that hybridize with a target nucleic acid underconditions that amplify the target sequence. Also, the sample geneticmaterial can be combined with a normal target nucleic acid sequence or anormal sequence probe (e.g., fluorescent or non-fluorescent) controlthat does not have any genetic variation so as to prepare a ratio of˜1:1, which is near equivalent test and control genetic material.However, it is possible to vary this ratio substantially, such as from1:10 to 10:1.

The normal target nucleic acid sequence or normal probe sequence (e.g.,control nucleic acid) are combined with the genetic material of thesample (e.g., sample nucleic acid), and then amplified in a singlereaction tube. Alternatively the control nucleic acid and sample nucleicacid can be mixed after separate amplification procedures. A controlnucleic acid of the normal target nucleic acid alone is also amplifiedsimultaneously (however, with improvements to distinguishing individualstrands of nucleic acids, it may be possible to run the control withinthe same reaction as the sample that is being interrogated). Thedenaturation profiles of the control nucleic acid and the sample nucleicacid can then be determined by high resolution melting curve analysis ofthe control and sample nucleic acids. Exemplary normal, or wild type,nucleic acid regions with known mutations that correspond to a change indrug resistance are listed in Table 3.

The denaturation profile data for these tests can be storedelectronically. As such, the control or sample data may be retrievedfrom a previous analysis so that it can be used for a comparison of theresults. The ability to save the denaturation profile data can eliminatethe need to always perform a control reaction with each run of the testsample. The data for samples is compared data for the normal targetcontrol, and any differences or variations between the two data sets arescored as a variation in the target region for the unknown sample. Whenthe sample includes a variation, the sample (i.e., microorganism) isclassified as being potentially resistant to the drug that targets thegenetic region (e.g., target nucleic acid sequence) that is the subjectof the test.

In some embodiments, a sample target nucleic acid (e.g. DNA or RNA) isprepared with control target nucleic acid so as to obtain a mixture ofsample and control target nucleic acid at about a 1:1 ratio. This can beachieved by mixing the sample and control nucleic acids, orco-amplifying the sample and control nucleic acids (e.g., by PCR) atabout a 1:1 ratio of starting material. These sample and control nucleicacids are initially denatured at a temperature high enough to ensure thesample target nucleic acids and the normal control target nucleic acidsare all denatured. The nucleic acids in the mixture (e.g., sample andcontrol) are then annealed at some temperature below the meltingtemperature where they begin to denature (e.g., Tm). For example, theannealing temperature can be 10° C. or more below the Tm of the targetcontrol nucleic acid. The mixture is then subjected to slow heating, andthe amount of hybridized sample and control nucleic acids present in thetube are monitored. The monitoring can be performed by fluorescence ofthe double-stranded nucleic acid product, wherein the fluorescence isgenerated by the inclusion of a dye which binds only to double-strandednucleic acids. The dye can be included in an amount that saturates thetemplate. The fluorescent signal is lost as the double-stranded nucleicacids begin to denature, and less sites are available for binding to thesaturating dye. The denaturation procedure is continued until nodouble-stranded nucleic acid is present, and the fluorescence is nearlyzero. The fluorescent data obtained during the denaturation procedure isthen saved for computing and comparing against control denaturation datathat is prepared with a similar protocol using only the control targetnucleic acid. As such, a high resolution melting curve analysis can beperformed with the mixture of the sample and control nucleic acids andthe composition having only the control nucleic acids, and a comparisoncan be made between the two melting curves. A difference between themelting curves can be an indication that the sample nucleic acids arefrom a microorganism that has drug resistance to the drug that interactswith the target nucleic acid or gene product thereof.

In some embodiments, any protocol or instrument that can distinguishbetween the hybridized sample and control nucleic acids from thedenatured sample and control nucleic acids can be used. The denaturationdata obtained from the sample denaturation curves that were generatedfrom the mixture having the sample and control nucleic acids arecompared to denaturation data of the control nucleic acid. Thedenaturation data of the control nucleic acid can be either storedcontrol denaturation data or the control nucleic acid can be denaturedand monitored in a separate reaction chamber along with the experimentalsample. The melting profiles of the normal control target are comparedwith the experimental sample so that any differences in these meltingprofiles can indicate the presence of a variation in the target region.When the control is a normal control target nucleic acid, variations inthe sequences can indicate the microorganism is resistant to the drugthat interacts with the target nucleic acid or gene product thereof.

In some embodiments, the co-amplified sequences of enriched MTb DNA andcontrol MTb DNA are simultaneously denatured, and then annealed toproduce homoduplexes of amplified control MTb DNA and enriched MTb DNA,and also produce heteroduplexes of the control and enriched MTb DNAs. Asaturating double-stranded DNA binding dye, such as a dye thatfluoresces when interacting with a duplexed nucleic acid, is included inthe amplification mixture to enable the generation of high resolutionmelting curve data from these homoduplexes and heteroduplexes. As such,the annealed samples of homoduplexes and heteroduplexes as well as thecontrol MTb DNA are subjected to high resolution melting curve analysisthat is monitored using fluorescence or other method of detecting thebinding dye.

The data obtained from monitoring the high resolution melt of thehomoduplexes, heteroduplexes, and control MTb DNA are input into acomputing system so that computing methods can be employed to analyzethe data. A mathematical comparison of the control MTb DNA sample datawithout added enriched sample DNA is then computed against the samplecontaining the co-amplified homoduplexes and heteroduplexes. Themathematical comparison, after normalization of the curves bytemperature and beginning and ending points, allows the subtraction ofeach data point along the melting curve of the sample containing theco-amplified product from the control MTb DNA sample data. The resultinggraph for invariant samples that have sequences that are notsubstantially different from the control MTb DNA is essentially a flatline with minor variation about zero. A graph for samples that haveheteroduplex DNA (e.g., control DNA with enriched sample DNA) thatcontains base pairing mismatches will show a change in the meltingcurve, and when subjected to the subtraction algorithm will produce adistinctly different graph than the flat graph of control and invariantsequences.

Samples that contain variant graphs from the control sample graphs arescored as variant in the drug target region (e.g., nucleic acid target),and microorganisms are likely to be less susceptible (e.g., resistant)to the action of the drug for this genetic region. Also, several drugtarget nucleic acid regions can be amplified simultaneously in differentreaction chambers for a single patient or for multiple patients.

FIGS. 6A-6C provide illustrations that show results of methods of highresolution melting curve profiles for determining the presence of avariation in a sample target nucleic acid sequence from a normal targetnucleic acid sequence. The presence of the variation is an indicationthat the microorganism is resistant to a drug, such as rifampicin. Moreparticularly, FIG. 6A depicts the hybridization products, either by PCRamplification or alternative template enrichment method, of normal(e.g., non-resistant strains nucleic acids) and resistant strains. Thenormal template (e.g., control target nucleic acid) is included in themixture with the sample target nucleic acid to produce an imperfectmatch between the nucleic acids that are hybridizing FIG. 6B showsmelting curves that have slight differences between the two meltingcurves, which are differences in melting profiles of the control targetnucleic acid and the mixture with the sample target nucleic acid. FIG.6C shows a difference in the melting curves between the control and thesample. The normal control target nucleic acid profile is plotted as thesolid line sample, which has no difference from the “normal” nucleicacid of microorganisms that are sensitive to the drug. The dashed lineshows a distinct difference between the “normal” and the mis-matchedsample, which indicates the microorganism is a resistant strain.

FIG. 7 is a graphical representation of high resolution melting curveanalysis between +/− control nucleic acid, nucleic acids from aresistant strain, and nucleic acids from a strain that is sensitive tothe drug. The graph was prepared using an automated curve differencecalling software (Idaho Technology, LightScanner), and shows the abilityto distinguish resistant samples from sensitive samples. Any samplewhich is called, by the software, as the same as the negative control issensitive to the drug, and any sample called as different from thenegative control is classified as resistant to the drug. The analysispackage can be configured in any arrangement desired. Alternatively, anymethod that can graphically represent the difference between the shapesof the curves, especially in the upper region of the curve, can be usedto differentiate between the ‘normal’ sequence and the test sequencepotentially containing a mismatch. Further, the differences can beobserved directly from the melting curves without further analysis.

In some embodiments, the high resolution melting curve analysis may beused in any genetic test for the detection of variation or similaritiesbetween sample nucleic acids and normal control nucleic acids.

In some embodiments, the amplification and/or denaturation can be usedto screen for normal samples by using various altered probes instead ofprobes of a normal sequence.

In some embodiments, the amplification and/or denaturation can be toscreen for mutated, non-normal, target nucleic acids using properlydesigned altered probes.

In some embodiments, the amplification and/or denaturation can be usedfor detecting commonalities between samples, such as forensicidentification testing.

In some embodiments, the amplification and/or denaturation can be usedfor epidemiological surveying of different samples.

In some embodiments, the probes used in the amplification may be eitherDNA or RNA (e.g., natural, or synthetic, or from amplified sources).

In some embodiments, the amplification and/or denaturing can be used toconfirm the presence of wild type sequences.

In some embodiments, the amplification and/or denaturing can be used toconfirm the presence of wild type sequences to further demonstrate thatthe test sample comes from an organism that will be sensitive to thedrug represented by that region.

In some embodiments, the amplification can be performed with real-timeor conventional PCR methods. Also, any amplification method can be usedthat will produce sufficient quantities of normal control nucleic acidsand/or target region genetic material to allow detection by aninstrument with suitable detection capabilities.

In some embodiments, the denaturation or melting curve analysisdetection system may be any high resolution melting instrument or anappropriately adapted instrument capable of generating sufficientresolution with basic sample heating and detection capabilities.

In some embodiments, the normal and sample nucleic acids can beamplified by PCR in a single tube. Alternatively, the normal and samplenucleic acids can be amplified in separate tubes and then mixed prior tothe high resolution melting curve analysis.

In some embodiments, the normal nucleic acids may be retrieved fromstock solutions, and then mixed with the amplified sample nucleic acidsin appropriate ratios to generate similar results.

In some embodiments, the normal nucleic acids that are used as thecontrol can be distinguished from mixtures with the sample nucleic acidsby using a variety of chemistries in order to produce an internalcontrol. This can include the use of different chemistries, such asusing fluorescently labeled normal control nucleic acids. As such, onlyduplexes that are formed from the labeled control nucleic acids cangenerate a fluorescent melting signal, which is specific to the normalcontrol template.

In some embodiments, the sample nucleic acids and the control nucleicacids may be compared after both the sample and control are amplifiedand/or denatured in different runs, which could be on different days.

In some embodiments, the high resolution melting curve analysis can beperformed on any instrument capable of denaturing and/or annealingnucleic acids, and capable of detecting the amount of hybridized nucleicacids compared to the denatured nucleic acids.

In some embodiments, sufficient instrument sensitivity can allow for theanalysis of the samples as described herein without having to amplifythe sample nucleic acids. That is, the instrument has sufficientsensitivity so that the sample nucleic acids are detectable withoutamplification.

In some embodiments, the analysis of the sample nucleic acids isperformed with high resolution annealing that monitors the nucleic acidsas they anneal. In part, this is possible because the annealing oftarget nucleic acids of the sample and control can be used as the meansto identify differences between the control template and the testsamples rather than only using the melting curve analysis ordenaturation.

In some embodiments, the present invention can study DNA and/or RNA froma microbiological or other biological sample for genetic variations. Thenucleic acids can be intact, fragmented, or portions of the entireorganism nucleic acid or the target region of the nucleic acid. Theprimers can be selected from a region of or adjacent to the portion ofthe target nucleic acid that is to be interrogated. The primers can benon-fluorescent, fluorescent, or capable of producing either anelectrostatic or electrochemical signal.

The amplification compositions can include the following: polymerasechain reaction ingredients, include reverse transcriptase, and/or DNApolymerase; appropriate buffers, salts, and deoxynucleotide and/ordeoxyribonucleotide triphosphates to amplify the target sequence; afluorescent double-stranded DNA binding dye, fluorescent probe,fluorescence resonance energy transfer probes, or other similar probemay be used to detect the formation of the annealed versus the denaturedRNA, or DNA/RNA homoduplex and/or heteroduplexes; oligonucleotideprimers designed to specifically amplify the target region of the samplenucleic acid and the normal control nucleic acid, wherein the primerscan be phosphodiester oligonucleotides, LNA oligonucleotides, PNAoligonucleotides, or any combination of these.

The instruments that can be used for the analysis of the sample nucleicacid can be any instrument capable of detecting the formation anddissolution of DNA/DNA, RNA/RNA, or DNA/RNA duplexes, and in furtherembodiments, DNA/protein or RNA/protein duplexes, or DNAhomotriplexes//homoquadruplexes. Such an instrument should be capable ofgenerating strong fluorescent signals when the targets are annealed andmonitor the change in fluorescence as the target nucleic acids denature.The instrument data can be recorded in a computing system havingsoftware configured for performing data analysis. Also, the instrumentcan be configured to perform both the nucleic acid amplification and thehybridization/denaturation. However, it is possible to perform thesefunctions in several distinct instruments without any detriment to theresults. An alternate configuration would be an instrument that couldmonitor the annealed and denatured status of the target sequences byultraviolet light, electrochemical signal generation, solutionviscosity, or other as yet undeveloped techniques.

The data obtained from the analysis of amplification and thehybridization/denaturation can be analyzed with any software packageconfigured to determine the differences between data. For example, asoftware package, currently available from Idaho Technology, or CorbettResearch, and soon from Roche Applied Science, that is designed tocompare the melting profiles of a normal target from those of thesamples where the normal target is hybridized can be used to identify nochanges, or minor or major differences. The exact format of the softwareoutput is unimportant; however, the software must simply be able toidentify those samples which have variations from normal melting curveprofiles compared to those that are normal.

The following Exemplary Aspects of specific aspects for carrying out thepresent invention are offered for illustrative purposes only, and arenot intended to limit the scope of the present invention in any way.

EXEMPLARY ASPECTS Example 1 Amplification and Analysis of DrugSensitivity Regions of Mycobacterium Tuberculosis

A. Whole Genome Amplification of M. Tuberculosis Genomic DNA

If sample DNA quantities are insufficient to obtain an amplified productfrom a drug sensitive region, then whole genome enrichment may be usedto amplify the sample DNA before amplification of specific regions, oramplicons. The whole genome enrichment in the MycoBuffer sample wasperformed in the parallel with an identical starting copy number oftemplate DNA that was suspended in water alone. To monitor the overallwhole genome enrichment of the mycobacterium tuberculosis, 3 distincttarget regions of the mycobacterium genome were chosen to evaluate eachfor enrichment. A real-time PCR assay method was used to screen each ofthese distinct target regions for enrichment compared to the un-enrichedcontrol samples. The point on the X-axis that the sample line begins totrend upward is indicative of the quantity of starting genetic materialin the sample, the earlier on the X-axis that the signal begins tochange the higher the quantity of starting material.

FIG. 5A is a graph that shows the results obtained from the CFP32 targetregion of Mycobacterium tuberculosis, which compares the 22 base randomprimer mixed with samples in water or MycoBuffer. The graph shows theMycoBuffer enriched sample trends upwards earlier and indicates a higheramount of starting material.

FIG. 5B is a graph that shows the results obtained from the IS6110target region of Mycobacterium tuberculosis, which compares the 22 baserandom primer mixed with samples in water or MycoBuffer. Again, thegraph shows the MycoBuffer enriched sample trends upwards earlier andindicates a higher amount of starting material.

FIG. 5C is a graph that shows the results obtained from the btMTb targetregion of Mycobacterium tuberculosis, which compares the 22 base randomprimer mixed with samples in water or MycoBuffer. Yet again, the graphshows the MycoBuffer enriched sample trends upwards earlier andindicates a higher amount of starting material.

B. Sample Preparation

Using the Petroff method of sputum sample sterilization, MTb wasseparated from human cellular material. Briefly, the Petroff method isas follows: the Petroff method, sputum was homogenized for 15 min in ashaker by using an equal volume of 4% sodium hydroxide. Aftercentrifugation at 3,000 rpm for 15 min in a Megafuge 1.0 (Heraeus), thedeposit was neutralized with about 20 ml of sterile distilled water.Samples were again centrifuged. The ‘deposit’ or pelleted material istransferred to a microcentrifuge tube and lysed with Mycobuffer(RAOGene, Inc, Milford, Pa.) according to the manufacturer'sinstructions. The supernatant material from this lysis protocol containsthe Mycobacterial DNA, and is transferred to a fresh microcentrifugetube.

C. Primary Screen for the Presence of M. Tuberculosis DNA in a Sample

Non-quantitative screening for Mycobacterial DNA in sample using theFluoresentric MTb Screen (HTPCR method) was performed by combining thefollowing ingredients in a 20 uL reaction, the products can be detectedby real-time PCR with probes, or electrochemical detection, or by gelelectrophoresis, or any other suitable method. Here we describe the useof real-time PCR to detect the product formation:

Combine:

10×FI15 Buffer: (500 mM Tris-HCl (pH 8.0); 5 mg/mL Bovine Serum Albumin(nonacetylated); 10 mM MgCl2, 40% Dimethylsulfoxide) use at 1× finalconcentration.

10× deoxynucleotide triphosphates (2 mM each) use at 1×.

10×LC Green I (Idaho Technology, Inc.) use at 0.5×.

Thermo-stable DNA polymerase (Tfi(exonuclease−) (Invitrogen Corp,Carlsbad, Calif.) use 2.5 U/reaction.

Oligonucleotide primers designed to have temperature overlap with thegene encoding the PPE Family Protein. Primers for amplified product:

(SEQ ID NO. 205) FI15-MTb FOR: CCGGAAACGTCGGCATCGCAAACTC (SEQ ID NO.206) FI15-MTb REV: TGCCCGTGTTGTAGAAGCCCGTGTTGAA

PPE Family gene

Add Mycobuffer/DNA sample to reaction.

Amplify according to the following protocol:

95 C denaturation: 1 min

75 C activation: 10 minutes

90 cycles of:

85 C for 30 s

75 C for 30 s with a fluorescent acquisition

1 Melt cycle:

95 C for 10 s

60 C for 10 s

Ramp to 95 C taking fluorescence acquisitions along the temperature rampto generate a melting curve of the product.

Final products may demonstrate variable melting profiles, as shown inFIG. 8. Those reactions that amplify a specific product are indicativeof the presence of MTb DNA in a test sample.

D. Alternative Detection Method for Amplified Product

Alternative to real-time PCR detection: electrochemical detection

Using the same basic primers with the following addenda:

FI15-MTb FOR: (SEQ ID NO. 205) Biotin-CCGGAAACGTCGGCATCGCAAACTC FI15-MTbREV: (SEQ ID NO. 206) Fluorescein-TGCCCGTGTTGTAGAAGCCCGTGTTGAA

Perform amplification as above. Remove sample and performelectrochemical detection according to manufacturer's directions(Anzenbio). Briefly, place in electrochemical detector chip (AnzenBio),incubate 20 minutes. The neutravidin chip binds to the biotin on theforward (or reverse primer . . . depending on the ultimate design) andthe chip is washed with 1× Phosphate buffered saline+1% Tween 20 (PBST),3 times. Add anti-fluorescein antibody conjugated to Horse RadishPeroxidase, incubate 20 minutes. Wash plate 3× with 1×PBST, add TMB(electrochemical detection buffer) and incubate 1 minute. Measure signalformation with PSD-8 detector. Signals in excess of 5 are scored aspositive, those less than 5 are scored as negative. A reference negativesample, and positive sample should be included to confirm these results.

Alternative detection, the products can be visualized by gelelectrophoresis, any product formation other than those seen in thenegative control sample should be considered suspect of being positivefor MTb.

Alternative detection, using capillary gel electrophoresis. Same as gelelectrophoresis.

Alternative detection, HPLC, Mass Spectroscopy, Spectroscopy,Fluorimetry, and the like. Detection of the presence of an amplified PCRproduct in a sample can be achieved using any available techniques,preferably those that can differentiate amplified products by size asopposed to just quantity. The presence of an amplified product,especially one in the expected size range, is indicative of the presenceof Mycobacterium tuberculosis (MTb) in a test sample.

E. Molecular Enrichment by Whole Genome Amplification

Molecular Enrichment Protocol:

Although most samples tested to date have had sufficient DNA for directamplifications of target drug sensitivity regions, some samples of MTbDNA will contain very small quantities of DNA for use in the MTb DrugResistance Screen. To overcome this problem a basic technique to enrichthe samples using a modified whole genome amplification procedure hasbeen employed. Basically, the samples are subjected to the followingprotocol:

DNA solution in Mycobuffer is added to random oligonucleotides. Thesolution is denatured and cooled to room temperature (allows randombinding of oligonucleotides throughout the genome of the MTb). Thesolution is then mixed with whole genome amplification mixture andincubated for 8 hours to produce a whole genome enrichment, on averagethe genome is enriched 100× over the raw sample.

DNA incubation solution is:

1-5 uL of DNA

1× Thermopol buffer (New England Biolabs)

100 uM random 22-mer primers (final concentration).

In a total volume of 15 uL

Denature for 2 minutes at 96 C

Cool to Room Temp for 10 minutes

Place on ice

Add 35 uL of molecular enrichment mix (whole genome amplification mix)

Final concentrations of ingredients are:

400 uM dNTPs

1× Thermopol buffer

0.35 U/uL of BST thermo stable polymerase (Bacillus stearothermophillus)large fragment (exonuclease—)

4% Dimethyl sulfoxide

T4 gene 32 protein (30 ng/ul)

Incubate 8 hrs @ 50 C.

Incubate 15 minutes @ 80 C

Hold at 4 C until use.

Samples are then purified by a modified filter binding assay. Briefly,this method can be employed at any stage in the process where DNA is tobe separated from the solution a quick method to buffer and primerexchange the DNA. We have found the Direct binding of the Mycobuffersolution+Binding Mix works well for earlier stage purification is neededor desired.

Sample DNA in Mycobuffer is mixed 1:1 with Binding Mix (4M GuanidiniumHCl, 12.5 mM Tris-HCl, pH 6.8, 0.5% NP40 detergent, 50% Ethanol)

Samples are loaded onto a Whatman GF/F 1.0 um glass fiber filter 96 welllong drip filter plate, and incubated at room temperature for 2 minutes.The filter plate is stacked on a 96×2 mL waste collection plate.

Samples are centrifuged (1800 rpm for 10 minutes) or vacuum filtered(slowly), remove plates from centrifuge or vacuum system.

200 uL of Wash Buffer I (1.6 M Guanidinium HCl, 10 mM Tris-HCl, pH 6.8,and 0.1% NP40, 70% Ethanol) is added and centrifuged or vacuum filtered.Plates are removed from centrifuge or vacuum system.

400 uL of Wash Buffer II (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 60%Ethanol) is centrifuged or vacuumed. Followed by a second wash with 200uL Wash Buffer II,

Collection reservoir is emptied, and entire system is either vacuumdried or dried by air, at RT for 2 hrs, or at 56 C for 20 minutes.

A fresh collection plate is stacked with filter plate, and 100 uL ofElution Buffer (DNase, RNase Free sterile water) is added to thefilters. Samples incubate at RT for 2 minutes, and are centrifuged, orvacuum filtered.

This entire system is a modification of existing methods using these 96well filter plates.

Sample DNA is in the eluate. Alternatively, the amplified genomeproducts can be used directly without purification for furtheramplification procedures if the genomic reaction contents and randomprimers don't interfere with subsequent specific amplifications.

This material is used for downstream processes.

The sample DNA is then evaluated with QPCR to confirm the amount of DNApresent in the sample. This serves three purposes: 1 is to perform asecondary screen for the presence of MTb in the sample; 2 is to verifythe molecular enrichment; and 3 is to establish an overallquantification of the amount of DNA present in the sample. This methodas previously developed has demonstrated a consistency of enrichment ofno more than 3 fold variation in the enriched sample DNA. Our methoduses three genes to confirm the enrichment. One gene result is used foradditional downstream processing.

F. Real Time Amplification of Samples Containing M. Tuberculosis DNA

Each sample is subjected to real-time PCR quantification of the enrichedsample DNA. Using a fluorigenic probe system (5′ nuclease assay, TaqMan)as the signal generating moiety.

The reaction components are as follow:

Reaction Mix:

10×PCR buffer (500 mM Tris-HCl, pH 8.5, 5 mg/ml BSA (non-acetylated), 40mM MgCl2,)

10×dNTP 2 mM Each (also may use 4 mM dUTP for contamination clean uppurposes)

Enzyme (Thermostable DNA Polymerase) Tfi exonuclease+ (invitrogen,Carlsbad, Calif.)

And oligonucleotide primers (three different reactions possible persample), use primers at 0.5 uM final, and fluorigenic probes at 0.2 uMFinal.

Pan Mycobacterium Assay: MTb27.3 (conserved protein):

(SEQ ID NO. 207) CFP32 FOR: TCGTTCATCACCGATCC (SEQ ID NO. 208) CFP32REV: GTGAGCAGTTCGTTCCA (SEQ ID NO. 209) CFP32 TM:FLUORESCEIN-TCAACGAGACGGGCACGCT-BHQ1

IS6110 Transposase:

IS6110 FOR; (SEQ ID NO. 210) TGCGAGCGTAGGCGTC IS6110 REV: (SEQ ID NO.211) GTCCAGCGCCGCTTC IS6110 TM; (SEQ ID NO. 212)FLUORESCEIN-CTGCTACCCACAGCCGGTTAGGT-BHQ1

PPE Family Protein Gene:

(SEQ ID NO. 213) BTTb FOR; GCCAGCATTGAGGAT (SEQ ID NO. 214) BTTb REV;CAATTCGGGCACCAATAA (SEQ ID NO. 215) BTTb TM;FLUORESCEIN-TGCGATGCCGACGTTTCCG-BHQ1

IS6110 is a target that is not reliable for quantification as it ispresent in the genome of MTb>1/genome.

BTTb and cfp32 genes are used for establishing enrichment in the assay,both have similar signal crossing values at cycle 34, versus theunenriched samples which are either ‘flat” for BTTb or are 100× (>6cycles) later than the enriched sample (cfp32).

Each reaction has 1-5 uL of purified or prepared DNA solution added tothe final reaction mix. The volume information must be noted in thereaction setup, as the exact volume will become a numerical divisor fordownstream processing. It is important to determine the relativequantity of MTb DNA in a sample for subsequent mixing steps.

Amplification and Melting Analysis of MTb DNA Sample and Controls

The enriched sample DNA is then analyzed with the drug sensitivitymarker assays. This assay is based on the following information:hybridized DNA which is perfectly matched by Watson/Crick base pairingrules will generate a characteristic melting curve of the melting DNA.When the same sequence is hybridized with a similar sequence of DNA thatcontains 1 or more ‘mis-matched’ bases along the length, thecharacteristic melting curve is no longer generated, but rather a newcurve is generated that indicates the difference in the meltingcharacteristics for the mismatched strands of DNA. To generate themismatched sequences it is not simply sufficient to use the amplifiedDNA for the sample in question, rather it is necessary to also include asample of DNA that contains a sequence of DNA that is the ‘unmodified’DNA. Such that, when the two are mixed in nearly equal proportions priorto, during, or at the end of an amplification reaction, and they arehybridized together a significant percentage of the hybridized templatesare in ‘mis-matched’ hybridizations. Such that a melting curve can begenerated that will indicate the presence of a ‘mutation’ in the samplesequence. If these sample sequences that are being amplified aredesigned to surround the nucleic acid sequences of genes that are eitherthemselves or their gene products are the target of antimicrobial drugs.Then any aberrant melting behavior from the samples will make suspectthe use of a particular drug for the treatment of the microbiologicalinfection as the organisms DNA will indicate, or at least suggest, thatthe drug will be ineffective. This may potentially be applied to cancerchemotherapy, viral drug resistance, and antimicrobial drugs.

The process for performing this screen is as follows. Using the resultsfrom the three secondary screens for MTb following molecular enrichmentof the samples, the results from the Cfp32 sample are used to calculatethe amount of DNA present in the sample. Alternatively, any MTb ampliconcan be used to determine the quantity of DNA in the MTb sample relativeto control DNA as long as the amplicon is present in both. Further,separate control and unknown amplicons could be assessed for determiningthe relative DNA concentrations in control and test samples. Our purposefor using only one has been for ease of calculation.

The calculated crossing threshold from the Roche LightCycler 480instrument, or virtually any real-time PCR machine, is used to calculatethe concentration of ‘wild-type’ RPOB (Rifampicin) drug target to add tothe screening reaction. The control stock solution (1:1,000,000 dilutionof master stock) is diluted 10× for every 4 cycles that the samplecrosses baseline after cycle 18, in the case of the example above with aCT value of 32, this is 14 cycles or a 5000× dilution (10^3.69). Thiscan be easily presented as a chart for the user or as a simple piece ofsoftware that will calculate the volumes to be mixed prior toamplification and/or melting.

The sample DNA and an equal amount of RPOB ‘normal, wild type,unmutated, non-resistant DNA’ is added to the reaction. The reactionconsists of the following components:

10×PCR buffer (500 mM Tris-HCl, pH 8.5, 5 mg/ml BSA, 30 mM MgCl2,)

10×dNTP mix (No dUTPs)

Oligonucleotides at 0.5 uM (final) each

LCGreen+ or LCGreen Dye (Idaho Technology, Inc)

Enzyme (Tfi (exo+)) or other thermostable polymerase with proofreadingactivity.

Mycobacterium RpoB gene, target of Rifampicin (Antibiotic):

RPOB FOR: CAAGGAGTTCTTCGGCACC (SEQ ID NO. 13) RPOB REV:GGACCTCCAGCCCGGCA (SEQ ID NO. 14)

A control reaction with only the RPOB sample as well as 1,2 or more‘resistant’ controls can and should also be performed simultaneously, inseparate reactions. We have two control reactions where we have mixed inequal proportions the RPOB normal control with one of the following: 1 asingle point mutation in the target region, or 2 a 3 base deletion ofthe target region. These three samples serve to ensure the assay isperforming as expected, controls for each drug target should be includedand would essentially have similar characteristics.

The samples are amplified by the following protocol, on a LightCycler480 instrument.

95 C for 10 minutes

40 cycles of:

95 C for 10 s

57 C for 10 s

72 C for 40 s

1 Cycle of Melting:

95 C for 10 s

50 C for 10 s

70 C for 30 s

95 C for 0 s with fluorescence acquisitions set to 25-35acquisitions/degree C. (High Resolution melting). The data can then beanalyzed using the soon to be released High Resolution Melting curvemodule for the LC 480 instrument or by using the LightScanner softwarefrom (Idaho Technology, Inc.). Both packages allow one to set thebaseline samples (the positive control samples, as standards). Further,any device that can measure the quantity of double stranded DNA (dsDNA)at specific temperatures during the melting can be used to generatemelting curves. The default curve settings are usually sufficient,though occasionally the settings must be modified to be sure that thecontrol samples are being accurately called. If control samples areaccurately called then the reaction results can be deemed acceptable andthe diagnostic call can be made. Thus, a difference between the controlwild-type melting curve and a melting curve from an unknown sample isindicative of a point mutation or polymorphism between the samples. Inthis case, with the rpoB region of MTb, the difference between meltingcurves is indicative of the presence of Rifampicin resistant DNA in atest sample, and thus can be used to diagnose the presence of Rifampicinresistant MTb in a sample. In a similar manner, this technique can beapplied to analyze any DNA region where there are known mutations thatcorrelate with a change in a phenotype, and is especially powerful forthe assessment of drug resistance or sensitivity.

Example 2 Determination of Drug Resistance or Sensitivity in Human MTBSamples

The purpose of these experiments was to demonstrate that clinicalsamples previously tested and confirmed to contain MTb could be rapidlyassessed for drug resistance or sensitivity. Blinded clinical samplesfrom MTb patients were obtained that had been prepared by the Petroffmethod and were resuspended in MGIT buffer (Becton Dickinson). Sampleswere assessed for Rifampicin and Streptomycin resistance using primerpairs, amplicons and melting temperatures listed in Tables 2 and 3.

MTb Test Protocol:

Run samples against H37RV standard sample using cfp32 Taqman assay toquantitate samples.

Mastermix: 1× Kappa without Sybr buffer (Kappa Biosystems SYBRG1 mastermix without SYBR), 1 ul cfp32 oligo, 1.75 mM MgCl, QS to 9 ul with water

Place 18 ul of mastermix per sample into 384 well plate.

Add 2 ul of samples.

Place a plate seal on plate and spin plate

Run in LC480 under cfp32 run protocol.

Denature 10 min at 95 C

Amplify: 95 C for 10 sec, 59 C for 40 sec (50 cycles)

From cfp32 assay determine dilution factor needed for samples using theequation C=S*E^N

where C=10000000, E=Standard curve efficiency, N=Cp value

Dilute samples to lowest concentration sample in Myco buffer.

Run diluted samples in 80 bp rpob assay to determine resistance.

Mastermix: 1× Kappa without Sybr, 0.5 ul 180 bp Oligo, 1× Eva green dye,QS to 9 ul with water

Place 9 ul of mastermix per sample into 384 well plate.

Add samples 0.5 ul of H37RV+0.5 ul of samples into well.

Place a plate seal on plate and spin down

Run in LC480 under rpob run protocol.

Denature: 10 min at 95 C

amplification: 95 C for 10 sec

57 C for 10 sec

72 C for 40 sec with single acquisition

Run samples until all samples have reached plateau for 2 or 3 cycles(approx. 30 cycles).

End amplification protocol and all samples to go through meltingprotocol.

Remove plate from LC480 and centrifuge to collect any condensation fromtop.

Melting protocol ramp to 95 C for 1 sec

50 C for 1 sec

70 C for 30 sec Start collecting melt data continuously at 30acquisitions/degree C.

End data collection at 95 C

Melt samples on HR-1 instrument.

Move samples from 384 well plate to 20 ul capillary tubes.

Briefly spin labelled capillaries in centrifuge to collect samples atbottom.

Using HR-1 instrument control software, melt samples individually usingthe FI LAB MTb Opt Melt protocol.

Ramp rate 0.07

acquisition start at 80 C with target Fluorescence of 90%

End acquisition at 96 C

Cool to 40 C

Note: Run 2 samples prior to data collection to allow instrument to warmup properly.

Data Analysis

Open up HR-1 Melt Analysis tool software.

Open folder containing data files and click “select current directory”

Select samples to analyze and click “continue”

Under “analyze” select normalize

Adjust left two cursors to approximately one degree before melt begins

Adjust left two cursors to approximately one degree after melt ends

Click OK

Under “Analyze” select temperature shift.

Under samples select a wild type sample to standardize to.

Adjust cursors to magnify melt region.

Select OK

Under “Analyze” select difference plot

Select wild type sample to standardize to.

Move cursors to select region of interest

click OK

Samples showing peaks on a curve difference plot above or below afluorescence level of 1.5 to 2 is considered resistant.

FIG. 10 shows the results of this procedure in curve difference plotformats using primers from Table 2 for the amplification and thecorresponding amplicon from Table 3 for the annealing and meltinganalysis. FIG. 10A shows the analysis of 4 samples for rifampicinresistance or sensitivity along with the control (wt1). FIG. 10Bdemonstrates the ability to identify Streptomycin resistance in MTbsamples. These data demonstrate that this technique can successfullydifferentiate between regions of DNA that are correlated with Drugsensitivity and those containing polymorphisms correlated with DrugResistance. In a similar manner, the methods and reagents disclosedherein can be used to assess sensitivity or resistance to all of thefirst line antibiotics used to treat MTb infections.

Example 3 Determination of Resistance to Anti_Fungal Agents

Fungal and yeast infections are responsible for a large number ofdiseases in humans. Some brief examples of clinically significant fungiinclude:

Malassezia furfur and Exophiala werneckii (superficial skin)

Piedraia hortae and Trichosporon beigelii (hair)

Microsporum species, (skin and hair)

Epidermophyton species (skin and nails)

Trichophyton species (skin, hair, and nails)

Sporothrix schenckii, Cladosporium species, Phialophora species, andFonsecaea species (subcutaneous/lymphatic tissues—chromoblastomycosis)

Histoplasma capsulatum, Coccidioides immitis, Fusarium species,Penicillium species (systemic respiratory)

Blastomyces dermatitidis (subcutaneous/respiratory)

Cryptococcus neoformans (respiratory/CNS)

Aspergillus species, Mucor species, Candida species, and Rhizopusspecies (opportunistic involving various body sites)

Further, fungi are often responsible for common infections such as yeastinfections, jock itch, athletes foot, and other dermatological issues.Resistance to antifungal agents will make treatment ineffective, soidentification of appropriate drugs is useful.

In order to determine the resistance pattern to terbinafine, a commonantifungal agent used for Saccharomyces and Candida infections, primersfrom Table 2 were used to amplify the corresponding amplicon from Table3 using the method presented in Example 2. DNA isolated from wild-typeS. cerevisiae or a template containing a mutation in the ERG1 gene thatconfers terbinafine resistance were used as the starting materials, andthe results of the melting curve difference analysis are shown in FIG.11. The mutated sequence is easily discerned from the wild typesequence. Thus, this method can be used to determine drug resistance orsensitivity in fungal infections as well as other pathogenic infections.

Example 4 Determination of Resistance and Sensitivity Patterns for HumanCancers to Chemotherapeutic Taxanes

Taxanes such as taxol, paclitaxel and docetaxel are potentchemotherapeutic agents used to treat wide varieties of cancers. Theirmechanism of action is shared by epithilones and work by binding andstabilizing tubulin polymers in cells. The binding sites for these drugson tubulin has been described (Rao, S., Orr, G. A., Chaudhary, A. G.,Kingston, D. G., and Horwitz, S. B. (1995) J. Biol. Chem.270:20235-20238) and mutations in this region or beta tubulin can causeresistance to taxanes (Table 3). Template DNA was purified by standardmeans and subjected to the method presented in Example 2, using primersfrom Table 2 to amplify the corresponding regions in Table 3. Twoamplicons with mutations that caused resistance to taxanes (B-tub R282Qand B-tub T247I) were easily distinguishable from two independentreactions with wild type DNA (wt1 and wt2; FIG. 12) using thismethodology. Thus, this method could be used to diagnose sensitivity orresistance to chemotherapeutic agents to allow physicians theopportunity to better understand the nature of the cancer and whattreatments are likely to be effective or ineffective.

Example 5

Malaria is an infectious disease caused by the parasite calledPlasmodia. There are four identified species of this parasite causinghuman malaria, namely, Plasmodium vivax, P. falciparum, P. ovale and P.malariae. 300-500 Million people are infected each year. The most commontreatment is chloroquine, but resistance to chloroquine has beenemerging. Currently, the World Health Organization (WHO) utilizes amethod to detect chloroquine resistant mutations that takes 28 days.

The method described in Example 2 was similarly applied to determine itsability to differentiate between chloroquine resistant and chloroquinesensitive DNA. The primers presented in Table 2 were used to amplify theamplicon presented in Table 3. FIG. 13 shows the results of this assay,which demonstrate that the method can readily identify a mutation inthis region that results in Chloroquine resistance versus a normalchloroquine sensitive region. Thus, this method could be used to assessthe drug sensitivity of parasite infections and allow better treatments.Further, this assay can be performed in less than a day, which issignificantly faster than current methods(http://www.malariasite.com/MALARIA/DrugResistance.htm).

Example 6 Determination of Drug Resistance in HIV Infected Individuals

Zidovudine (INN) or azidothymidine (AZT) (also called ZDV) is anantiretroviral drug, the first approved for treatment of HIV. Itsmechanism of action is through blockage of the HIV reversetranscriptase, which prevents replication of the viral genetic material.Mutations in regions of the HIV reverse transcriptase have rendered theviruses resistant to these first line drugs.

FIG. 14 shows the melting curve difference plots of 2 independent runsusing the method presented in Example 2 to discriminate between wildtype and ZDV-resistant DNA. The primers presented in Table 2 were usedto amplify the regions presented in Table 3. This example clearlydemonstrates that this method is applicable to determining drugresistance or sensitivity in viral pathogens as well.

Example 7 Determination of Methicillin Resistance in StaphylococcusAureus Infections

Methicillin-resistant Staphylococcus aureus (MRSA) infection is causedby Staphylococcus aureus bacteria—often called “staph.” Decades ago, astrain of staph emerged in hospitals that was resistant to thebroad-spectrum antibiotics commonly used to treat it. Dubbedmethicillin-resistant Staphylococcus aureus (MRSA), it was one of thefirst germs to outwit all but the most powerful drugs. MRSA infectioncan be fatal. Because of this, it is important to determine whether agiven staph infection is multi drug resistant so that proper treatmentcan be administered. Generally, staph is collected from tissues or nasalsecretions, but can also be isolated from throat samples or open wounds.Standard methods are used to extract the DNA from the clinical sample,

FIG. 15 demonstrates that this method can discriminate between multidrug resistant staph infections and normal staph infections. Using theprimers presented in Table 2 to amplify the region disclosed in Table 3,with the method presented in Example 2, this method could discernbetween wild type regions of the staph DNA and regions with a singlepoint mutation that results in multi drug resistance.

Example 8 Assessment of MTB Infection by Dynamic Flux Amplification

Biological samples suspected of being infected with MTb were assessedfor the presence of MTb DNA using dynamic flux amplification. Humansamples either positive or negative for MTb infection were treated usingthe following procedure:

Oligos are the FI-15 MTb primers (Example 1)

The reaction conditions are:

1: 10×FI-15 Buffer (50 mM Tris-HCl, 8.0; 0.25 mg/ml Native(non-acetylated BSA); 2 mM MgCl₂; 4% DMSO; 2 mM each dNTPs)

2: Enzyme Gene-Choice HS-TaqPolymerase, although other thermostable DNApolymerases are acceptable.

3: Primers at 0.5 uM Final

4: Thermal cycling: 90-10 sec

Either 74, 76, 78, or 80° C.-10 sec (50+ cycles) (currently done in<1.25 hrs)

FIG. 16 shows the results of this reaction at different temperatures forthe thermocycling. This experiment was performed in a PCR to simulatethe conditions of a heating block in the lab, which when set at 80° C.displayed a temperature cycle of +/−5° C. Each pair of wells performedat a single temperature contains a first reaction that uses a templatepositive for MTb DNA, and the second a template negative for MTb DNAprepared similarly. The expected 150 bp amplification product appears atall temperature cycling conditions tested only in samples positive forMTb, but is not amplified in control samples. Thus, a field DNAamplification test could be used to assess MTb infections in humansamples, using only a standard sample collection and preparationprotocol, a heating block to amplify a specific product, and a means todetect said product. This has the potential to allow field diagnosis ofMTb infection without the need to send the samples to a designatedtesting center. Further, it can give a rapid result, requiring only alittle over an hour of thermocycling time to amplify the product.

Example 9 Dynamic Flux Amplification to Identify the Presence ofSalmonella Typhimurium in a Test Sample

The purpose of this example is to demonstrate that dynamic fluxamplification can be used to amplify a specific region of DNA from abiological sample. Thus, instead of using a costly PCR machine, suchreactions could take place in a heating block or any device that holds atemperature. If the hold is not highly accurate and maintains thetemperature through cycling between heating and off phases, there is anatural flux in the temperature. This is true for heating blocks,heating ovens, and even refrigerators or freezers (although coolinginstead of heating).

Salmonella typhimurium DNA was isolated from biological samples bystandard methods. Samples or control DNA (no template or E. colitemplate) mixtures were prepared and subjected to the followingconditions:

Forward primer: caccacgctcaccgatgatgccctgctttg Tm 77C Reverser primer:actgggagccattaaccgcatcggtgctg Tm 75C

Template:

Tm = 92C actgggagccattaaccgcatcggtgctgtccgcggccagggtgcctgccgccagattggtgattttgctggcgcttccgttacggctggcgctgaatgtgccagaggctgcatcccaaagcagggcatcatcggtgagcgtggtg

Reagents:

10× buffer (same as before)

dNTPS (2 mM each)

3 mM MgCl

Primers @ 0.5 uM each

Dye: LC Green (Idaho Technology, Inc Salt Lake City, Utah)

Enzyme: Tfi (exo−) Invitrogen

Thermal cycling conditions: initial hold at 79 C for 15 minutes(equivalent to the use of a heating block set to 77-78 degrees Celsius);90 Cycles: 79 1 min; 76 1 min FIG. 17 shows that a specific product isamplified detectable at cycle 62 and higher. Amplification is only seenin the reaction containing S. typhimurium DNA and not in samplescontaining no DNA or E. coli DNA (not shown). Thus, this technologycould be used to identify the presence of S. typhimurium in a biologicalsample and indication the presence of bacterial infection if the sampleis of non-bacterial origin, such as a human sputum sample or throatswab. Advantageously, the above method can amplify DNA without the useof a thermocycler. Detection of amplified products can be assessed byany traditional methods, including, but not limited to, gel analysis orelectrophoresis, UV detection, fluorescent detection, gold detection,capture of hybrids in a ELISA or rapid in vitro diagnostic assay,capture of amplified products by lateral flow, and the like. In someembodiments, primers may be labeled, especially at the 5′ end or withinternal labels, to allow detection of specific amplified products.

REFERENCES

The following U.S. patents and Pre-Grant Publications are eachincorporated herein by specific reference; U.S. Pat. Nos. 4,683,195;4,965,188; 6,214,587; 6,692,918; 5,219,727; 5,476,774; 5,314,809;6,040,166; 6,197,563; 6,001,611; 6,617,137; 7,074,600; 6,977,148;6,740,745; 6,033,881; 7,160,998; 7,081,226; 20070054311; 20050233363;20050181394; 20040248105; and 20070020672.

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety for all purposes asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference for allpurposes.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications can be made thereto without departing from the spiritor scope of the appended claims.

The invention claimed is:
 1. A method for nucleic acid sequenceamplification, comprising: identifying a target nucleic acid sequencefrom one or more segments of DNA, said target nucleic acid sequencehaving primers with melting temperatures that are within at least 15° C.of the melting temperature of the target nucleic acid sequence;obtaining a pair of oligonucleotide primers with an annealing curve(T_(A)) that overlaps with a denaturation curve (T_(D)) of the targetnucleic acid sequence, in such a manner as to minimize the temperaturerange between the higher of the melting temperature of a pair ofoligonucleotide primers and the melting temperature of the targetnucleic acid sequence, wherein the temperature range does not exceed 15°C.; and amplifying the target nucleic acid sequence by thermocycling thetarget nucleic acid sequence and the pair of oligonucleotide primerswithin a 15° C. temperature range, wherein thermocycling comprises: i.denaturing the target nucleic acid sequence; and ii. annealing of thepair of oligonucleotide primers; and iii. extension of the targetnucleic acid sequence by the pair of oligonucleotide primers, wherein anupper thermal cycle temperature in the thermocycling is selected tominimize non target denaturation and maximize target denaturation. 2.The method of claim 1, wherein the temperature range between the meltingtemperature of one of the pair of oligonucleotide primers and themelting temperature of the target nucleic acid sequence is not greaterthan 2.5° C., wherein the one of the pair of oligonucleotide primers hasa greater melting temperature range difference with the meltingtemperature of the target nucleic acid sequence than the second of thepair of oligonucleotide primers.
 3. The method of claim 1, wherein thetemperature range between the higher of the melting temperature of apair of oligonucleotide primers and the melting temperature of thetarget nucleic acid sequence is 82° C. to 89° C.
 4. The method of claim1, wherein the melting temperature of at least one of theoligonucleotide primers is the same as the melting temperature of thetarget nucleic acid sequence.
 5. The method of claim 1, wherein themelting temperature of at least one of the oligonucleotide primers iswithin 2.5° C. of the melting temperature of the target nucleic acidsequence.
 6. The method of claim 1, wherein annealing and denaturing ofthe oligonucleotide primers to the target nucleic acid sequence occur ina dynamic flux.
 7. The method of claim 1, wherein amplifying producesspecific amplification products of the target nucleic acid sequence. 8.The method of claim 1, wherein amplifying decreases the formation ofnon-specific amplification products compared with amplifying within abroad temperature range.
 9. The method of claim 1, wherein the amplifiedtarget nucleic acid sequence is detected by a colorimetric detectionreadout.
 10. The method of claim 9, wherein the readout is a positive ornegative signal.
 11. The method of claim 1, further comprising detectingthe amplified target nucleic acid sequence by fluorescence emitted froma DNA-binding fluorescent dye.
 12. The method of claim 1, furthercomprising assaying by high resolution melting curve analysis theamplified target nucleic acid sequence for sequence variations relativeto a reference sequence.
 13. The method of claim 12, wherein the meltingcurve analysis detects one or more base pair matches between theamplified target nucleic acid sequence and the reference sequence. 14.The method of claim 1, wherein the one or more segments of DNA are froman infectious microorganism and wherein the target nucleic acid sequencecomprises at least one polymorphism that is associated withresponsiveness of the microorganism to a drug.
 15. The method of claim14, further comprising identifying a reference sequence that includes atleast one polymorphism associated with responsiveness to the drug. 16.The method of claim 14, wherein the at least one polymorphism isassociated with sensitivity to the drug.
 17. The method of claim 15,wherein the at least one polymorphism of the reference sequenceindicates sensitivity to the drug.
 18. The method of claim 14, whereinthe at least one polymorphism is associated with resistance to the drug.19. The method of claim 15, wherein the at least one polymorphism of thereference sequence indicates resistance to the drug.
 20. The method ofclaim 1, wherein the target nucleic acid sequence includes at least onepolymorphism.
 21. The method of claim 20, further comprising assayingthe amplified target nucleic acid sequence for sequence variationsrelative to a reference sequence.
 22. The method of claim 1, wherein thethermocycling is carried out within a temperature range that is within 2degrees of the temperature range between the melting temperature of theoligonucleotide primers and the melting temperature of the targetnucleic acid sequence.
 23. The method of claim 1, wherein thethermocycling is carried out within the temperature range that isbetween the melting temperature of the oligonucleotide primers and themelting temperature of the target nucleic acid sequence.
 24. The methodof claim 1, wherein the temperature range between the meltingtemperature of one of the oligonucleotide primers and the meltingtemperature of the target nucleic acid sequence is not greater than 5°C., wherein the one of the oligonucleotide primers has the greatestmelting temperature difference with the melting temperature of thetarget nucleic acid sequence.
 25. The method of claim 1, wherein thetemperature range between the melting temperature of one of theoligonucleotide primers and the melting temperature of the targetnucleic acid sequence is not greater than 10° C., wherein the one of theoligonucleotide primers has the greatest melting temperature differencewith the melting temperature of the target nucleic acid sequence. 26.The method of claim 1, wherein the melting temperature of at least oneof the oligonucleotide primers is within 5° C. of the meltingtemperature of the target nucleic acid sequence.
 27. The method of claim1, wherein the melting temperature of at least one of theoligonucleotide primers is within 10° C. of the melting temperature ofthe target nucleic acid sequence.
 28. A method for rapid amplificationof a target nucleic acid sequence, comprising: identifying a targetnucleic acid sequence from one or more segments of DNA; determining themelting temperature of the target nucleic acid sequence via an iterativeprocess comprising amplifying the target nucleic acid sequence and thendetermining the melting temperature of the target nucleic acid sequencevia melting curve analysis; and generating a pair of oligonucleotideprimers having an annealing curve (T_(A)) that overlaps with adenaturation curve (T_(D)) of the target nucleic acid sequence, in sucha manner as to minimize the temperature range between the meltingtemperatures (Tm) of the pair of oligonucleotide primers and the meltingtemperature (Tm) of the target nucleic acid sequences; and amplifyingthe target nucleic acid sequence by thermocycling the target nucleicacid sequence and the pair of oligonucleotide primers within a 15° C.temperature range, wherein thermocycling comprises: i. denaturing thetarget nucleic acid sequence; and ii. annealing of the pair ofoligonucleotide primers; and iii. extension of the target nucleic acidsequence by the pair of oligonucleotide primers.
 29. The method of claim28, wherein the melting temperatures (Tm) of the pair of oligonucleotideprimers are within 2.5° C. of the melting temperature (Tm) of the targetnucleic acid sequence.
 30. The method of claim 28, wherein the meltingtemperatures (Tm) of the pair of oligonucleotide primers are within 5°C. of the melting temperature (Tm) of the target nucleic acid sequence.31. The method of claim 28, wherein the melting temperatures (Tm) of thepair of oligonucleotide primers are within 10° C. of the meltingtemperature (Tm) of the target nucleic acid sequence.
 32. The method ofclaim 28, wherein the melting temperatures (Tm) of the pair ofoligonucleotide primers are within 15° C. of the melting temperature(Tm) of the target nucleic acid sequence.
 33. The method of claim 28,wherein the melting temperatures (Tm) of the pair of oligonucleotideprimers and the melting temperature (Tm) of the target nucleic acidsequence are between 75° C. and 90° C.
 34. The method of claim 28,wherein the melting temperatures (Tm) of the pair of oligonucleotideprimers and the melting temperature (Tm) of the target nucleic acidsequence are between 82° C. and 89° C.
 35. The method of claim 28,further comprising assaying a resulting amplicon for sequence variationsrelative to a reference sequence.
 36. The method of claim 28, furthercomprising adjusting the pH of the amplification reaction to increasethe specificity of selectively amplifying the target nucleic acid overother nucleic acids.
 37. A dynamic flux method of nucleic acid sequenceamplification, comprising: a. combining a pair of forward and reverseoligonucleotide primers with a target nucleic acid sequence to beamplified; and b. amplifying the target nucleic acid sequence bythermocycling the pair of forward and reverse oligonucleotide primersand the target nucleic acid sequence within a 15° C. temperature rangethat is defined by the area contained within the overlap of an annealingcurve (T_(A)) of the pair of oligonucleotide primers and thedenaturation curve (T_(D)) of the target nucleic acid sequence, whereineach forward and reverse oligonucleotide primer has a meltingtemperature (T_(m)) within 15° C. of the T_(m) of the target nucleicacid sequence, and wherein thermocycling comprises: i. denaturing thetarget nucleic acid sequence; and ii. annealing of the forward andreverse oligonucleotide primers; and iii. extension of the targetnucleic acid sequence by the forward and reverse oligonucleotideprimers.
 38. The dynamic flux method of claim 37, wherein amplifying thetarget nucleic acid sequence by thermocycling the pair of forward andreverse oligonucleotide primers and the target nucleic acid sequenceoccurs within a 10° C. temperature range.
 39. The dynamic flux method ofclaim 37, wherein amplifying the target nucleic acid sequence bythermocycling the pair of forward and reverse oligonucleotide primersand the target nucleic acid sequence occurs within a 5° C. temperaturerange.
 40. The dynamic flux method of claim 37, wherein amplifying thetarget nucleic acid sequence by thermocycling the pair of forward andreverse oligonucleotide primers and the target nucleic acid sequenceoccurs within a 2.5° C. temperature range.
 41. The method of claim 1,wherein each of the pair of oligonucleotide primers is present in anapproximately equimolar concentration.
 42. The method of claim 28,wherein each of the pair of oligonucleotide primers is present in anapproximately equimolar concentration.
 43. The method of claim 37,wherein each of the forward and reverse oligonucleotide primers ispresent in an approximately equimolar concentration.